Monoclonal antibodies for enhancing or inhibiting insulin-like growth factor-I

ABSTRACT

The present invention provides αVβ3 integrin cysteine loop domain agonists and antagonists (including peptide agonists and antagonists and analogs thereof), along with methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/474,294, filed May 17, 2012, now allowed, which is a continuation ofU.S. application Ser. No. 12/498,719, filed Jul. 7, 2009, now U.S. Pat.No. 8,187,595, issued May 29, 2012, which is a continuation-in-part ofU.S. application Ser. No. 11/123,290, filed May 6, 2005, now U.S. Pat.No. 7,723,483, issued May 25, 2010, which claims the benefit of thefiling date of U.S. Application No. 60/657,151, filed Feb. 28, 2005, andU.S. Application No. 60/569,147, filed May 7, 2004. The disclosures ofthese applications are hereby incorporated by reference herein in theirentireties.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numberAG-02331 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

This invention describes methods for inhibiting or enhancing the actionsof insulin-like growth factor-I (IGF-I).

BACKGROUND OF THE INVENTION

IGF-I is a small polypeptide hormone that stimulates the growth of alltypes of cells. Because IGF-I has a broad spectrum of action andstimulates balanced tissue growth it has been implicated in thedevelopment of several important human cancers and also inatherosclerosis. IGF-I acts primarily on anchorage dependent cells thatare contained in these tissues. These cells also possess a class ofreceptors termed integrin receptors which are responsible for theirattachment to extracellular matrix molecules. In order for cells todivide normally, in response to extracellular stimuli the cell has tosense that its integrin receptors are bound to extracellular matrixmolecules. Therefore manipulation of ligand occupancy of integrinreceptors can alter processes that are important in disease developmentsuch as cell division and migration.

Our studies have determined that IGF-I stimulates endothelial and smoothmuscle cell division. They have further determined that these cellsutilize the αVβ3 integrin receptor to communicate to the cell nucleusthat they are adhered adequately to extracellular matrix in order todivide. The abundance of one specific integrin (the αVβ3 integrin) isrelatively restricted in human tissues and it is expressed primarily ingrowing cells and particularly in cells involved in the maintenance ofthe vasculature such as smooth muscle and endothelial cells. Our studieshave shown that occupancy of this integrin receptor with its naturallyoccurring ligands such as osteopontin, vitronectin and thrombospondin isrequired for these cells to respond to IGF-I with increased DNAsynthesis and cell migration. Blocking ligand occupancy of this integrinwith disintegrin antagonists results in inhibition of cell growth andmigration. Our studies have shown that this cooperative interactionbetween αVβ3 and the IGF-I receptor is mediated by regulating thetranslocation of two specific signaling molecules. These moleculesare 1) a protein tyrosine phosphatase termed SHP-2 and 2) a signalingprotein termed Shc. Under normal circumstances SHP-2 is localized in thecytoskeleton and cytosolic compartments of the cell. Following ligandoccupancy of αVβ3 the cytoplasmic domain of the β3 integrin undergoestyrosine phosphorylation. SHP-2 is transferred to the cell membrane bybinding to proteins that bind to the phosphorylated tyrosine residues inβ3. This transfer is necessary in order to localize SHP-2 to themembrane where it recruits other important signaling molecules such asShc. SHP-2 colocalization with Shc and/or dephosphorylation of signalingmolecules within the IGF-I signaling pathway is required for theiractivation and for subsequent transmission of signals from the IGF-Ireceptor to nucleus. Activation of the two major intracellular signalingpathways that are required for IGF-I activation (e.g. the PI-3 kinaseand MAP kinase pathways) can be inhibited by inhibiting either SHP-2 orShc transfer to the membrane. The site of localization of SHP-2 and Shcis a membrane protein termed SHPS-1. SHPS-1 is phosphorylated inresponse to IGF-I. This phosphorylation is required for SHP-2 and forShc transfer. Shc is phosphorylated after transfer to SHPS-1. BlockingαVβ3 ligand occupancy blocks both SHP-2 and Shc transfer thus inhibitingIGF-I stimulated cell growth.

Although methods have been described previously for inhibiting ligandoccupancy of the αVβ3 integrin, they all utilize a technology thatinhibits binding to a specific binding site on the αVβ3 heterodimer thatbinds to the arginine, glycine, asparagine (RGD) sequence within the ECMligands. Binding αVβ3 antagonists to this site is associated with drugtoxicity and side effects. Accordingly there is a need for new ways toinhibit, or activate, IGF-1 actions that do not utilize the αVβ3 bindingsite that binds to the RGD sequence.

SUMMARY OF THE INVENTION

In our invention we have determined that there is a second binding siteon αVβ3 that binds to several extracellular matrix proteins. Moreimportantly we have determined that enhancing ligand occupancy of thisdomain augments IGF-I signaling and inhibiting ligand occupancy of thisspecific domain, inhibits IGF-I actions. Importantly ligand occupancy ofthis second binding site does not stimulate the specific biochemicalevents that are stimulated by peptides that bind to the RGD bindingsite.

A first aspect of the present invention is an αVβ3 integrinextracellular matrix protein binding site (or cysteine loop domaincontained in amino acids 177-184 of the β3 subunit) antagonist (e.g., apeptide antagonist or analog thereof or antibody that binds the cysteineloop domain).

A particular embodiment of the foregoing is an antibody thatspecifically binds to the αVβ3 integrin extracellular matrix proteinbinding site (or cysteine loop domain) (e.g., specifically binds to thecysteine loop domain at amino acids 177 to 184 of a human β3 integrin;optionally but preferably does not specifically bind the RGD bindingsite of a human β3 integrin; and optionally but preferably specificallybinds to the cysteine loop domain at amino acids 177 to 184 of a pig β3integrin. In some embodiments, the antibody is (a) the monoclonalantibody produced by hybridoma LAM-CLOOP-101, or (b) a monoclonalantibody that competes for binding to the same epitope as the epitopebound by a monoclonal antibody produced by the hybridoma LAM-CLOOP-101(i.e., a monoclonal antibody that specifically binds to the epitopebound by a monoclonal antibody produced by the hybridoma LAM-CLOOP-101.

A second aspect of the present invention is an αVβ3 integrinextracellular matrix protein binding site (or cysteine loop domain)agonist (e.g., a peptide agonist or analog thereof).

A further aspect of the present invention is a pharmaceuticalformulation comprising an active agent as described herein in apharmaceutically acceptable carrier.

A further aspect of the present invention is a method of inhibitingIGF-1 actions in a subject in need thereof, comprising administeringsaid subject an αVβ3 integrin extracellular matrix protein binding site(or cysteine loop domain) antagonist in an amount effective to inhibitIGF-1 actions in said subject. For example, the subject may be afflictedwith a tumor (e.g., breast cancer tumors, colon cancer tumors, lungcancer tumors, and prostate cancer tumors, and the antagonistadministered in an amount effective to treat the tumor. In someembodiments, the tumor or blood vessels supplying the tumor expressesαVβ3 receptors.

In another example, the subject is afflicted with atherosclerosis (e.g.,coronary atherosclerosis), and the antagonist is administered in anamount effective to treat the atherosclerosis. In some embodiments theatherosclerosis is characterized by atherosclerotic lesion cells thatexpress αVβ3 receptors. In another example the subject is afflicted withosteoporosis, and the antagonist is administered in an amount effectiveto treat the osteoporosis.

In another example the subject is afflicted with pathologicalangiogenesis (e.g., vascularization of a tumor, including tumors that doexpress levels of integrin αVβ3 detectable by immunohistochemistry andtumors that do not express levels of integrin αVβ3 detectable byimmunohistochemistry), and the antagonist is administered in an amounteffective to treat the pathological angiogenesis.

In another example, the subject is afflicted with diabetes (e.g., type Idiabetes, type II diabetes, diabetic retinopathy, diabetic nephropathy),and the antagonist is administered in an amount effective to treat thesecomplications of diabetes.

An aspect of the invention is, in a method of treating a tumor in asubject in need thereof by administering a treatment effective amount ofan antineoplastic compound or radiation therapy to the subject, theimprovement comprising administering to the subject an αVβ3 integrincysteine loop domain antagonist in an amount effective to inhibit IGF-Iaction in the subject (e.g., and thereby enhance the activity of theantineoplastic compound or radiation therapy to the subject, inhibitbone loss in the subject, or both) Subjects may be afflicted with tumorssuch as breast cancer tumors, colon cancer tumors, lung cancer tumors,and prostate cancer tumors. In some embodiments the tumor expresses αVβ3receptors.

A still further aspect of the present invention is a method of enhancingIGF-1 action in a subject in need thereof, comprising administering saidsubject an αVβ3 integrin cysteine loop domain agonist in an amounteffective to enhance IGF-1 action in the subject. For example, thesubject (e.g., infant, juvenile or adolescent subjects) may be afflictedwith insufficient growth, and the agonist administered in an amounteffective to enhance the growth of the subject. In another example thesubject (e.g., an infant subject) is afflicted with defective retinalvascularization, and the agonist is administered in an amount effectiveto treat the defective retinal vascularization. In another example, thesubject is afflicted with an ischemic injury (e.g., peripheral vasculardisease with claudication, myocardial infarction, etc.), and the agonistis administered in an amount effective to treat the ischemic injury. Inanother embodiment the subject is afflicted with neuronal atrophy orfailure of neural process development, and the agonist is administeredin an amount effective to treat the neuronal atrophy or facilitateneural process development. In another embodiment the subject (e.g., anadult or geriatric subject) is afflicted with a hip fracture and theagonist is administered in an amount effective to treat the hipfracture. In another embodiment the subject is afflicted with a diabeticischemic ulcer, and the agonist is administered in an amount effectiveto treat the diabetic ischemic ulcer.

A further aspect of the present invention is the use of an active agentas described herein for the manufacture of a medicament for carrying outa method of treatment as described herein.

A further aspect of the invention is a computer-based method foridentifying compounds that modulate activity of IGF-1, comprising: (a)providing a plurality of coordinates (e.g., at least 20, 30 or 40coordinates) for the cysteine loop domain (amino acids 177-184) of anαVβ3 integrin in a computer; (b) providing a structure of a candidatecompound to the computer in computer readable form; and (c) determiningwhether or not the candidate compound fits into or docks with a bindingcavity of the cysteine loop domain, wherein a candidate compound thatfits or docks into the binding cavity is determined to be likely tomodulate activity of IGF-1.

A further aspect of the invention computer-based method for rationallydesigning a compound that modulates activity of IGF-1, comprising: (a)generating a computer readable model of an extracellular matrix proteinbinding site of an αVβ3 integrin; and then (b) designing in a computerwith the model a compound having a structure and a charge distributioncompatible with the binding site, the compound having a functional groupthat interacts with the binding site to modulate acetyl-CoA carboxylaseactivity.

In one embodiment the present invention provides a method of screeningcompounds for activity in modulating cellular activation by IGF-1,comprising the steps of: (a) contacting, preferably in vitro, a testcompound to a system comprising a β3 integrin; then (b) determiningwhether the test compound binds to the cysteine loop at amino acids 177to 184 of the β3 integrin; and then (c) identifying the test compound asactive in modulating cellular activation by IGF-1 if the compound bindsthe cysteine loop domain. In some embodiments the test system comprisesαVβ3 integrin as a complex. The determining step may be carried out byany suitable means, such as by determining whether or not the testcompound inhibits the binding of an antibody, agonist or antagonist asdescribed herein that specifically binds to the cysteine loop domain.The integrin is preferably a mammalian β3 integrin, such as human or pigβ3 integrin.

Another embodiment of the present invention provides a method ofscreening compounds for activity in modulating cellular activation byIGF-1, comprising the steps of: (a) contacting, preferably in vitro, atest compound to a cysteine loop domain, wherein the cysteine loopdomain is a peptide comprising amino acids 177 to 184 of a β3 integrin;then (b) determining whether the test compound binds to the cysteineloop domain; (c) identifying the test compound as active in activatingcellular activation by IGF-1 if the compound binds the cysteine loopdomain. The peptide may consist of not more than 8, 10, 15 or 20 aminoacids, and includes amino acids 177-184 of the β3 integrin in sequence.In some embodiments the cysteine loop domain is in solution; in someembodiments the cysteine loop domain is immobilized on a solid support(e.g., as an affinity column). The determining step may be carried outby determining whether or not the test compound inhibits the binding ofan antibody, agonist or antagonist as described herein that specificallybinds to the cysteine loop domain. Again the integrin is preferably amammalian β3 integrin such as human or pig β3 integrin. In someembodiments the peptide comprising amino acids 177 to 184 of a β3integrin.

The present invention is explained in greater detail in the followingnon-limiting Examples.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

In general, the present invention encompasses a technology tospecifically inhibit ligand occupancy of the αVβ3 integrin through analternative binding site that does not lead to activation of specificintracellular signaling events that can lead to drug toxicity.Administration of antagonists that inhibit the binding of vitronectin tothis alternative αVβ3 binding site has been shown to block IGF-Istimulated activation of PI-3 kinase, MAP kinase, DNA synthesis and cellmigration. All of these events are important for IGF-I to stimulatesmooth muscle cell growth within atherosclerotic lesions. Similarlyintestinal smooth muscle cells express this integrin so inhibiting IGF-Iactions in this cell type could be useful in the treatment ofinflammatory bowel disease. We have also shown that this technology isuseful for inhibiting ligand binding to this site on αVβ3 that isexpressed on the surface of endothelial cells therefore antagonists thatinhibit ligand binding will likely inhibit IGF-I signaling and thereforecould be effective treatments of diabetic retinopathy and forangiogenesis that is associated with tumor formation. The inventioninvolves the development of compounds that inhibit binding and canfunction as competitive antagonists for binding of ECM ligands that bindto this binding site on the αVβ3 integrin.

This invention helps address two major problems in drug development thathave inhibited progress in this field. The first problem concerns theIGF-I receptor. While monoclonal antibodies have been developed thatinhibit ligand binding to the 1GF-I receptor, the molecular radius ofthe binding site on the receptor is large therefore inhibiting ligandbinding to the IGF-I receptor is a difficult problem in drug developmentbecause of the size of the molecule that will be necessary to fullyinhibit binding. This invention in contrast inhibits binding to a verysmall binding site on the αVβ3 integrin. The actual binding site on theintegrin itself is encompassed by 8 amino acids and the minimum peptidewhich is effective inhibiting binding is 8 amino acids therefore themolecular radius of the binding site is substantially smaller than theligand binding site to the IGF-I receptor and it is of a size that smallmolecular weight antagonists can be developed. A second problem withantagonizing the IGF-I receptor is that it is ubiquitously present onall cells. Therefore if a strategy were formulated to inhibit IGF-Ireceptor activity and this were used in combination with therapies thatstimulate apoptosis (e.g. a cancer chemotherapeutic or anantiangiogenesis drug) inhibiting IGF-I action in normal cells couldalso be associated with extensive apoptosis of normal cell types such asGI epithelium, bone marrow precursor cells and neurons. Therefore thetoxicity of a coadministered agent would be greatly amplified. Similarlyadministering IGF-I receptor antagonist even without a coadministeredagent is likely to lead to inhibition of protein synthesis and possiblyto apoptosis in normal cell types. In contrast this drug willselectively target the αVβ3 integrin. Because αVβ3 integrins that signalcooperatively with the IGF-I receptor are present on vascularendothelial and smooth muscle cells and are usually only expressed inhigh concentrations in proliferating cells, the compound being developedis quite selective since it specifically targets these cell types. Celltypes such as GI epithelium and bone marrow precursor cells which do notexpress abundant αVβ3 integrin will likely be spared toxicity. Thereforethe invention solves the problem of being able to develop low molecularweight inhibitors that inhibit IGF-I action. Secondly it addresses amajor problem of generalized toxicity that would be apparent with anyanti-IGF-I receptor antagonists. Third, it addresses the problem ofinhibiting the IGF-I receptor tyrosine kinase which leads to inhibitionof the insulin receptor tyrosine kinase and the development of diabetes.

Enhancing IGF-I action provides beneficial effects in several diseasestates. Certain neurologic diseases, such as amyotrophic lateralsclerosis have been shown to be partially responsive to IGF-I. Similarlysince glucose toxicity in neurons is inhibited by IGF-I then diseasessuch as diabetic neuropathy may be able to be treated with agents thatenhance ligand occupancy of αVβ3 since this is at times expressed onglial cells which provide trophic support for regenerating neurons. Itis also possible that a small molecule agonist given with IGF-I wouldstimulate wound healing or endothelial cell growth within vasculargrafts.

A. Definitions

Subjects that may be treated by the present invention include both humansubjects for medical purposes and animal subjects for veterinary anddrug screening and development purposes. Other suitable animal subjectsare, in general, mammalian subjects such as primates, bovines, ovines,caprines, porcines, equines, felines, canines, lagomorphs, rodents(e.g., rats and mice), etc. Human subjects are the most preferred. Humansubjects include fetal, neonatal, infant, juvenile and adult subjects.

Amino acid as used herein refers to a compound having a free carboxylgroup and a free unsubstituted amino group on the α carbon, which may bejoined by peptide bonds to form a peptide active agent as describedherein. Amino acids may be standard or non-standard, natural orsynthetic, with examples (and their abbreviations) including but notlimited to:

Asp=D=Aspartic Acid

Ala=A=Alanine

Arg=R=Arginine

Asn=N=Asparagine

Cys=C=Cysteine

Gly=G=Glycine

Glu=E=Glutamic Acid

Gln=Q=Glutamine

His=H=Histidine

Ile=I=Isoleucine

Leu=L=Leucine

Lys=K=Lysine

Met=M=Methionine

Phe=F=Phenylalanine

Pro=P=Proline

Ser=S=Serine

Thr=T=Threonine

Trp=W=Tryptophan

Tyr=Y=Tyrosine

Val=V=Valine

Orn=Ornithine

Nal=2-napthylalanine

Nva=Norvaline

Nle=Norleucine

Thi=2-thienylalanine

Pcp=4-chlorophenylalanine

Bth=3-benzothienyalanine

Bip=4,4′-biphenylalanine

Tic=tetrahydroisoquinoline-3-carboxylic acid

Aib=aminoisobutyric acid

Anb=α-aminonormalbutyric acid

Dip=2,2-diphenylalanine

Thz=4-Thiazolylalanine

All peptide sequences mentioned herein are written according to theusual convention whereby the N-terminal amino acid is on the left andthe C-terminal amino acid is on the right. A short line (or no line)between two amino acid residues indicates a peptide bond.

“Basic amino acid” refers to any amino acid that is positively chargedat a pH of 6.0, including but not limited to R, K, and H.

“Aromatic amino acid” refers to any amino acid that has an aromaticgroup in the side-chain coupled to the alpha carbon, including but notlimited to F, Y, W, and H.

“Hydrophobic amino acid” refers to any amino acid that has a hydrophobicside chain coupled to the alpha carbon, including but not limited to I,L, V, M, F, W and C, most preferably I, L, and V.

“Neutral amino acid” refers to a non-charged amino acid, such as M, F,W, C and A.

“αVβ3 integrin cysteine loop domain” as used herein refers to a specificregion on the αVβ3 integrin receptor (particularly mammalian receptors,e.g., those found endogenously in the subject being treated) that hasnot been identified previously as a region that would result in receptoractivation, and specifically excludes the RGD binding domain. Agoniststhat bind in this region include those containing a region of sequencethat is commonly termed a heparin binding domain. In general thecysteine loop domain or region of αVβ3 is occurring between amino acidsat positions 177 and 183 within the β3 subunit. See, e.g., B. Vogel etal., A novel integrin specificity exemplified by binding of the alpha vbeta 5 integrin to the basic domain of the HIV Tat protein andvitronectin, J. Cell Biol. 121: 461-8 (1993).

“IGF-I” as used herein means insulin-like growth factor-I.

“Treat” as used herein refers to any type of treatment or preventionthat imparts a benefit to a subject afflicted with a disease or at riskof developing the disease, including improvement in the condition of thesubject (e.g., in one or more symptoms), delay in the progression of thedisease, delay the onset of symptoms or slow the progression ofsymptoms, etc. As such, the term “treatment” also includes prophylactictreatment of the subject to prevent the onset of symptoms. As usedherein, “treatment” and “prevention” are not necessarily meant to implycure or complete abolition of symptoms.” to any type of treatment thatimparts a benefit to a patient afflicted with a disease, includingimprovement in the condition of the patient (e.g., in one or moresymptoms), delay in the progression of the disease, etc.

“Treatment effective amount”, “amount effective to treat” or the like asused herein means an amount of the inventive antagonist sufficient toproduce a desirable effect upon a patient inflicted with cancer, tumors,atherosclerosis, retinopathy, diabetic neuropathy, or other undesirablemedical condition in which IGF-I is inducing abnormal cellular growth.This includes improvement in the condition of the patient (e.g., in oneor more symptoms), delay in the progression of the disease, etc.

“Pharmaceutically acceptable” as used herein means that the compound orcomposition is suitable for administration to a subject to achieve thetreatments described herein, without unduly deleterious side effects inlight of the severity of the disease and necessity of the treatment.

“Antibody” or “antibodies” as used herein refers to all types ofimmunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The term“immunoglobulin” includes the subtypes of these immunoglobulins, such asIgG₁, IgG₂, IgG₃, IgG₄, etc. Of these immunoglobulins, IgM and IgG arepreferred, and IgG is particularly preferred. The antibodies may be ofany species of origin, including (for example) mouse, rat, rabbit,horse, or human, or may be chimeric or humanized antibodies. The term“antibody” as used herein includes antibody fragments which retain thecapability of binding to a target antigen, for example, Fab, F(ab′)₂,and Fv fragments, and the corresponding fragments obtained fromantibodies other than IgG. Such fragments are also produced by knowntechniques. In some embodiments antibodies may be coupled to orconjugated to a detectable group or therapeutic group in accordance withknown techniques.

“Therapeutic group” means any suitable therapeutic group, including butnot limited to radionuclides, chemotherapeutic agents and cytotoxicagents.

“Radionuclide” as described herein may be any radionuclide suitable fordelivering a therapeutic dosage of radiation to a tumor or cancer cell,including but not limited to ²²⁷Ac, ²¹¹At, ¹³¹Ba, ⁷⁷Br, ¹⁰⁹Cd, ⁵¹Cr,⁶⁷Cu, ¹⁶⁵Dy, ¹⁵⁵Eu, ¹⁵³Gd, ¹⁹⁸Au, ¹⁶⁶Ho, ^(113m)In, ^(115m)In, ¹²³I,¹²⁵I, ¹³¹I, ¹⁸⁹Ir, ¹⁹¹Ir, ¹⁹²Ir, ¹⁹⁴Ir, ⁵²Fe, ⁵⁵Fe, ⁵⁹Fe, ¹⁷⁷Lu, ¹⁰⁹Pd,³²P, ²²⁶Ra, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ⁴⁶Sc, ⁴⁷Sc, ⁷²Se, ⁷⁵Se, ¹⁰⁵Ag, ⁸⁹Sr,³⁵S, ¹⁷⁷Ta, ¹¹⁷mSn, ¹²¹Sn, ¹⁶⁶Yb, ¹⁶⁹Yb, ⁹⁰Y, ²¹²Bi, ¹¹⁹Sb, ¹⁹⁷Hg, ⁹⁷Ru,¹⁰⁰Pd, ^(101m)Rh, and ²¹²Pb.

“Chemotherapeutic agent” as used herein includes but is not limited tomethotrexate, daunomycin, mitomycin, cisplatin, vincristine, epirubicin,fluorouracil, verapamil, cyclophosphamide, cytosine arabinoside,aminopterin, bleomycin, mitomycin C, democolcine, etoposide,mithramycin, chlorambucil, melphalan, daunorubicin, doxorubicin,tamoxifen, paclitaxel, vincristin, vinblastine, camptothecin,actinomycin D, and cytarabine

“Cytotoxic agent” as used herein includes but is not limited to ricin(or more particularly the ricin A chain), aclacinomycin, diphtheriatoxin. Monensin, Verrucarin A, Abrin, Vinca alkaloids, Tricothecenes,and Pseudomonas exotoxin A.

“Detectable group” as used herein includes any suitable detectablegroup, such as radiolabels (e.g. ³⁵S, ¹²⁵I, ¹³¹I, etc.), enzyme labels(e.g., horseradish peroxidase, alkaline phosphatase, etc.), fluorescentlabels (e.g., fluorescein, green fluorescent protein, etc.), etc., asused in accordance with known techniques.

“Modulator” as used herein refers to a compound that binds to theindicated binding site (e.g., specifically binds to the cysteine loop ofβ3) and is an agonist or antagonist, or binds to an adjacent site andthereby affects binding at the cysteine loop of β3 domain (e.g., anallosteric inhibitor).

Applicants specifically intend that all United States patent referencescited herein be incorporated herein by reference in their entirety.

B. Agonists and Antagonists

Agonists and antagonists that may be used as active agents in carryingout the present invention may be in the form of a variety of differentstructures, as described further below. In general, the agonist binds toa specific region on the αVβ3 integrin receptor that has not beenidentified previously as a region that would result in receptoractivation. All agonists that we have determined to bind in this regioncontain a region of sequence that is commonly termed a heparin bindingdomain. This heparin binding domain is present in 5 ligands that we havefound to date that bind to this region of αVβ3. The documentation thatthey bind to a specific region of αVβ3 has been provided by two types ofexperiments, as discussed in Example 1 below.

The essential ligand structure of a preferred group of agonists isgenerally comprised by 8 amino acids. The five ligands that have beenshown to bind are connective tissue growth factor, heparin bindingepidermal growth factor, vitronectin, osteopontin and insulin-likebinding protein-5 (IGFBP-5) (SEQ ID NO: 1). Each of these ligands hasthe following sequence: BBXXABBB (SEQ ID NO:2) (B=basic, A=aromatic,X=Any). Using mutagenesis of these peptides, we have conducted multipleexperiments to determine the residues that are underlined are absolutelyrequired for activity. Substitutions with alanine for these 3 basicresidues results in between 70 and 90% reduction of binding affinity ofthese synthetic peptides for this region of αVβ3. We have alsodetermined that synthetic peptides with this structure (i.e., the regionof vitronectin contains the following sequence ³⁶⁷KKQRFRHR³⁷⁴ (SEQ IDNO:3) results in full stimulation of β3 phosphorylation, SHP-2 transferto downstream signaling molecules and enhancement of activation of theIGF-I receptor in response to IGF-I. We have shown that syntheticpeptides bearing this structure have full biologic activity andpotentiate the effect of IGF-I on DNA synthesis, cell migration, andprotein synthesis. Mutagenesis of the single arginine to alanine atposition 374 in vitronectin results in not only a 70% decrease inbinding, but also corresponding decreases in biologic activity asassessed by the three parameters noted above. Similarly alaninemutations of the first two basic residues also result in loss ofbiologic activity.

With respect to antagonists, we have determined that leaving the firsttwo residues intact, that is leaving them both basic followed byaddition of a hydrophobic residue at position 374 or a phosphorylatedserine in position 374 results in competitive antagonist: that is thesepeptides retain some ability to bind to αVβ3 but do not activate β3phosphorylation and/or SHP-2 transfer and do not enhance IGF-Istimulated receptor phosphorylation or cell division. An antagonist canalso be prepared using sequences from other ligands that are known tobind to this β3 domain. For example if the heparin binding domain ofIGFBP-5 (SEQ ID NO: 1) is used and there is a substitution for lysine208 to alanine this peptide binds to β3 but inhibits IGF-I actions.These data are discussed in greater detail in Example 2 below.

More particular examples of agonist and antagonist active agents aregiven below.

Agonists.

Agonists that may be used to carry out the present invention include butare not limited to compounds of Formula I:

wherein:

A¹ is a basic amino acid or a phosphorylated serine;

A² is a basic amino acid;

A³ is any amino acid;

A⁴ is any amino acid;

A⁵ is an aromatic amino acid;

A⁶ is a basic amino acid;

A⁷ is a basic amino acid;

A⁸ is a basic amino acid or a phosphorylated serine;

X is a chain of 0-5 amino acids (any), inclusive, the N-terminal one ofwhich is optionally bonded to R¹ and R²;

Y is a chain of 0-4 amino acids, inclusive, the C-terminal one of whichis optionally bonded to R³ and R⁴;

R¹, R², R³, and R⁴ are present or absent and are each independentlyselected from the group consisting of H, C1-C12 alkyl (e.g., methyl),C6-C18 aryl (e.g., phenyl, naphthaleneacetyl), C1-C12 acyl (e.g.,formyl, acetyl, and myristoyl), C7-C18 aralkyl (e.g., benzyl), andC7-C18 alkaryl (e.g., p-methylphenyl);

or a pharmaceutically acceptable salt thereof.

Antagonists.

Antagonists that may be used to carry out the present invention includebut are not limited to compounds of Formula II:

wherein:

A¹¹ is a basic amino acid;

A¹² is a basic amino acid;

A¹³ is any amino acid;

A¹⁴ is any amino acid;

A¹⁵ is an aromatic amino acid;

A¹⁶ is a basic amino acid;

A¹⁷ is a basic amino acid;

A¹⁸ is a noncharged or neutral amino acid or a hydrophobic amino acid;

X′ is a chain of 0-5 amino acids (any), inclusive, the N-terminal one ofwhich is optionally bonded to R¹¹ and R¹²;

Y′ is a chain of 0-4 amino acids, inclusive, the C-terminal one of whichmay be phosphoserine or may be optionally bonded to R¹³ and R¹⁴;

R¹¹, R¹², R¹³, and R¹⁴ are present or absent and are each independentlyselected from the group consisting of H, C1-C12 alkyl (e.g., methyl),C6-C18 aryl (e.g., phenyl, naphthaleneacetyl), C1-C12 acyl (e.g.,formyl, acetyl, and myristoyl), C7-C18 aralkyl (e.g., benzyl), andC7-C18 alkaryl (e.g., p-methylphenyl);

or a pharmaceutically acceptable salt thereof.

In Formulas I and II herein, the symbols X, Y, Z, A¹, A², A¹², and thelike stand for an amino acid residue, i.e., ═N—CH(R)—CO— when it is atthe N-terminus, or —NH—CH(R)—CO—N═ when it is at C-terminus, or—NH—CH(R)—CO— when it is not at the N- or C-terminus, where R denotesthe side chain (or identifying group) of an amino acid or its residue.Also, when the amino acid residue is optically active, it is the L-formconfiguration that is intended unless the D-form is expresslydesignated.

Peptide agonists or antagonists of the present invention, such ascompounds of Formula I and Formula II above, are preferably from 8 aminoacids to 11, 14 or 20 amino acids in length, and may have a molecularweight of from 600, 800 or 900 up to about 1200 or 2000.

Making Peptides.

Peptides of the present invention may be made in accordance withtechniques known in the art. Using accepted techniques of chemicalsynthesis, the peptide may be built up either from the N-terminus or,more typically, the C-terminus using either single amino acids orpreformed peptides containing two or more amino acid residues.Particular techniques for synthesizing peptides include (a) classicalmethods in which peptides of increasing size are isolated before eachamino acid or preformed peptide addition, and (b) solid phase peptidesynthesis in which the peptide is built up attached to a resin such as aMerrifield resin. In these synthetic procedures, groups on the aminoacids will generally be in protected form using standard protectinggroups such as t-butoxycarbonyl. If necessary, these protecting groupsare cleaved once the synthesis is complete. Other modifications may beintroduced during or after the synthesis of the peptide. Peptides of thepresent invention may also be produced through recombinant DNAprocedures as are known in the art.

Pseudopeptide Bonds.

In yet another aspect, the invention features analogs of Formula I orFormula II having at least one pseudopeptide bond between amino acidresidues therein. By “pseudopeptide bond” it is meant that the carbonatom participating in the bond between two residues is reduced from acarbonyl carbon to a methylene carbon, i.e., CH₂—NH; or less preferablythat of CO—NH is replaced with any of CH₂—S, CH₂—CH₂, CH₂—O, or CH₂—CO.Preferably, the pseudopeptide bonds are located between one or moreamino acid residues. In addition, such pseudopeptide bond analogs can beused to form dimeric analogs as is described above. A detaileddiscussion of the chemistry of pseudopeptide bonds is given in Coy etal. (1988) Tetrahedron 44:835-841.

Analogs.

active agents include analogs of the compounds of Formula I and FormulaII described herein. An “analog” is a chemical compound similar instructure to a first compound, and having either a similar or oppositephysiologic action as the first compound. With particular reference tothe present invention, analogs are those compounds which, while nothaving the amino acid sequences of the corresponding protein or peptide,are capable of acting as agonists or antagonists in substantially likemanner to the active compounds described herein. Such analogs may bepeptide or non-peptide analogs.

In protein or peptide molecules which interact with a receptor(specifically, the αVβ3 cysteine loop domain) the interaction betweenthe protein or peptide and the receptor generally takes place atsurface-accessible sites in a stable three-dimensional molecule. Byarranging the critical binding site residues in an appropriateconformation, peptides analogs which mimic the essential surfacefeatures of the peptides described herein may be generated andsynthesized in accordance with known techniques. Methods for determiningpeptide three-dimensional structure and analogs thereto are known, andare sometimes referred to as “rational drug design techniques”. See,e.g., U.S. Pat. No. 4,833,092 to Geysen; U.S. Pat. No. 4,859,765 toNestor; U.S. Pat. No. 4,853,871 to Pantoliano; U.S. Pat. No. 4,863,857to Blalock; (applicants specifically intend that the disclosures of allU.S. Patent references cited herein be incorporated by reference hereinin their entirety). See also Waldrop, Science 247, 28029 (1990);Rossmann, Nature 333, 392 (1988); Weis et al., Nature 333, 426 (1988);James et al., Science 260, 1937 (1993) (development of benzodiazepinepeptidomimetic compounds based on the structure and function oftetrapeptide ligands).

Non-peptide mimetics of the agonists or antagonists of the presentinvention are also an aspect of this invention. Non-protein mimetics maybe generated in accordance with known techniques such as using computergraphic modeling to design non-peptide, organic molecules able toagonize of antagonize binding in like manner as the active agentsdescribed herein. See, e.g., Knight, BIO/Technology 8, 105 (1990);Itzstein et al, Nature 363, 418 (1993) (peptidomimetic inhibitors ofinfluenza virus enzyme, sialidase). Itzstein et al., Nature 363, 418(1993), modeled the crystal structure of the sialidase receptor proteinusing data from x-ray crystallography studies and developed an inhibitorthat would attach to active sites of the model; the use of nuclearmagnetic resonance (NMR) data for modeling is also known in the art andsuch techniques may be utilized in carrying out the instant invention.See also Lam et al., Science 263, 380 (1994) regarding the rationaldesign of bioavailable nonpeptide cyclic ureas that function as HIVprotease inhibitors. Lam et al. used information from x-ray crystalstructure studies of HIV protease inhibitor complexes to designnonpeptide inhibitors.

Hydrophilic Groups.

Active agents of the present invention may include hydrophilic groupscoupled thereto, particularly covalently coupled thereto, to facilitatedelivery thereof, or improve stability, in accordance with knowntechniques (e.g., to the N-terminus of the peptide). Suitablehydrophilic groups are typically polyols or polyalkylene oxide groups,including straight and branched-chain polyols, with particularlyexamples including but not limited to polypropylene glycol),polyethylene-polypropylene glycol or poly(ethylene glycol). Thehydrophilic groups may have a number average molecular weight of 20,000to 40,000 or 60,000. Suitable hydrophilic groups and the manner ofcoupling thereof are known and described in, for example, U.S. Pat. Nos.4,179,337; 5,681,811; 6,524,570; 6,656,906; 6,716,811; and 6,720,306.For example, peptide agonists or antagonists can be pegylated using asingle 40,000 molecular weight polyethylene glycol moiety that isattached to the peptide.

Pharmaceutical Salts.

The active compounds disclosed herein can, as noted above, be preparedand administered in the form of their pharmaceutically acceptable salts.Pharmaceutically acceptable salts are salts that retain the desiredbiological activity of the parent compound and do not impart excessivetoxicological effects. Examples of such salts are (a) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; and salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; (b)salts formed from elemental anions such as chlorine, bromine, andiodine, and (c) salts derived from bases, such as ammonium salts, alkalimetal salts such as those of sodium and potassium, alkaline earth metalsalts such as those of calcium and magnesium, and salts with organicbases such as dicyclohexylamine and N-methyl-D-glucamine.

C. Formulations and Administration

For administration in the methods of use described below, the activeagent will generally be mixed, prior to administration, with anon-toxic, pharmaceutically acceptable carrier substance (e.g. normalsaline or phosphate-buffered saline), and will be administered using anymedically appropriate procedure, e.g., parenteral administration (e.g.,injection) such as by intravenous or intra-arterial injection.

The active agents described above may be formulated for administrationin a pharmaceutical carrier in accordance with known techniques. See,e.g., Remington, The Science And Practice of Pharmacy (9^(th) Ed. 1995).In the manufacture of a pharmaceutical formulation according to theinvention, the active compound (including the physiologically acceptablesalts thereof) is typically admixed with, inter alia, an acceptablecarrier. The carrier must, of course, be acceptable in the sense ofbeing compatible with any other ingredients in the formulation and mustnot be deleterious to the patient. The carrier may be a liquid and ispreferably formulated with the compound as a unit-dose formulation whichmay contain from 0.01 or 0.5% to 95% or 99% by weight of the activecompound.

Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient.

The active agents may be administered by any medically appropriateprocedure, e.g., normal intravenous or intra-arterial administration. Incertain cases, direct administration to an atherosclerotic vessel may bedesired.

Active agents may be provided in lyophylized form in a sterile asepticcontainer or may be provided in a pharmaceutical formulation incombination with a pharmaceutically acceptable carrier, such as sterilepyrogen-free water or sterile pyrogen-free physiological salinesolution.

Dosage of the active agent for the methods of use described below willdepend, among other things, the condition of the subject, the particularcategory or type of cancer being treated, the route of administration,the nature of the therapeutic agent employed, and the sensitivity of thetumor to the particular therapeutic agent. For example, the dosage willtypically be about 1 to 10 micrograms per kilogram subject body weight.The specific dosage of the antibody is not critical, as long as it iseffective to result in some beneficial effects in some individualswithin an affected population. In general, the dosage may be as low asabout 0.05, 0.1, 0.5, 1, 5, 10, 20 or 50 micrograms per kilogram subjectbody weight, or lower, and as high as about 5, 10, 20, 50, 75 or 100micrograms per kilogram subject body weight, or even higher.

D. Methods of Use: Agonists

Agonist active agents of the present invention can be utilized for avariety of different diseases. One preferred target disease state wouldbe children with short stature who secrete normal amounts of growthhormone. These are children who upon diagnostic testing cannot be provento be growth hormone deficient but are short. There is a reasonableamount of evidence to show that they are relatively resistant to theactions of growth hormone and IGF-I. Therefore a factor that enhancesIGF-I action should enhance the growth of these children and since theirIGF-I secretion is intact, no other therapy should be required forgrowth optimization.

Another application is for subjects afflicted with defective retinalvascularization. Premature infants who are severely malnourished and donot synthesize adequate IGF-I particularly in the back of the retina.This results in delayed and maldeveloped vascularization of the retinawhich can result in blindness or extremely decreased visual acuity.There is a critical period between 26 and 29 weeks wherein IGF-I actionis required. Although the cause of defective IGF-I action has not beenfirmly established, a factor which enhances IGF-I action on retinalvascular endothelial cells should provide a therapeutic benefit sincethese cells bear αVβ3 receptors.

Another application is to facilitate the development of collateral bloodvessels following ischemic injury. Both peripheral vascular disease withclaudication and myocardial infarction are associated with thedevelopment of collateral blood vessels during the healing period. Theability to form collaterals greatly determines the ability to recoverfrom these ischemic insults. Although several factors such as vascularendothelial growth factor have been shown to enhance collateralizationnone has proven to be clinically efficacious in patients. A potentialuse of this compound would be to stimulate smooth muscle and endothelialcells during the process of collateral vessel development.

Another application involves the treatment or inhibition of neuronalatrophy and failure of neural process development. This appliesspecifically to degenerative dementias and to diabetic neuropathy.

Still another application is to treat hip fractures in the elderly.Infusion of high concentrations of IGF-I has been shown to acceleratehealing from hip fractures in the elderly. The availability of thisagent would enable to one to directly inject it with the fracture sitewith a peptide that is likely to enhance osteoblast activity in thepresence of adequate IGF-I. The agonist can be administered with orwithout IGF-I to improve hip fracture healing.

Another application is to treat diabetic ischemic ulcers. In diabeticanimal models, it has been shown that addition of IGF-I with one of itsbinding proteins to wounds results in improved wound healing. Since indiabetes IGF-I concentrations are low addition of this agonist withIGF-I to wounds is likely to result in better wound healing in thepresence of diabetic ulcerations. In diabetic neuropathy it has beenshown that neurons undergo rapid apoptosis and have poor axonaloutgrowth unless adequate IGF-I is present. Since αVβ3 receptors arepresent on glial cells that provide support for neurons it is possiblethat this agonist could enhance neuronal outgrowth and the developmentof axonal processes and connections in diabetic neuropathy. Inneurodegenerative diseases such as ALS and various dementias, IGF-I hasbeen shown to inhibit neuronal apoptosis therefore therapy with thisagent may be of some use in preventing these neurodegenerativeconditions.

E. Methods of Use: Antagonists

Antagonism of IGF-I action has been shown to block lesion formation andearly atherosclerotic lesion development. Administration of anantagonist that blocks this binding site on αVβ3 would antagonize theeffect of matrix proteins that are abundant in atherosclerotic lesionssuch as vitronectin, osteopontin and fibrinogen. To the extent thatheparin binding epidermal growth factor and connective tissue growthfactor are active in atherosclerotic lesion development the antagonistwould also act to inhibit their effects.

Another use of antagonists would be to treat inflammatory bowel disease.Intestinal smooth muscle cells express αVβ3 receptors and theirproliferation in these diseases leads to intestinal strictures.Therefore inhibiting their growth with an antagonist could lead toprevention of this complication.

Another use of antagonists of the invention is in the treatment ofosteoporosis. Osteoblasts do not express αVβ3 but it is expressed onosteoclasts which stimulate bone reabsorption. Therefore inhibition ofstimulation of ligand occupancy on osteoclasts should result inenhancement of bone formation through the use of antagonist. Severalproteins such as osteopontin are abundant in bone extracellular matrixand could be stimulating osteoclasts through this mechanism thereforeantagonism of their action may allow IGF-I to increase bone formationwithout increasing bone resorption.

Another use of antagonists is to treat states of abnormal angiogenesis.Angiogenesis is important in tumor development but it is as important inother pathophysiologic processes such as diabetic retinopathy. Sinceendothelial cells express abundant αVβ3 receptors antagonists thatinhibit the binding of endothelial growth factors such as vascularendothelial growth factor or heparin binding epidermal growth factor toαVβ3 through this heparin binding domain would be expected to lead toinhibition of angiogenesis therefore this antagonist is a useful drug inthese clinical conditions.

Another use of antagonists is to treat cancers or tumors, particularlythose that have αVβ3 receptors (e.g., Wilm's tumor, nephroblastoma,neuroblastoma). Although αVβ3 is not an abundant receptor on all tumorcells, several tumor cell types that express αVβ3 have been described.Approaches to date have generally targeted the RGD sequence in ligandsthat stimulate αVβ3 and used antagonists that are binding to this domainto inhibit αVβ3 actions. Our approach that is antagonizing the cysteineloop on αVβ3 provides a unique approach targeting this receptor asopposed to the RGD binding site and thus may have greater efficacyinhibiting the development of these tumors.

In the treatment of cancers or tumors the active agents of the presentinvention may optionally be administered in conjunction with other,different, cytotoxic agents such as chemotherapeutic or antineoplasticcompounds or radiation therapy useful in the treatment of the disordersor conditions described herein (e.g., chemotherapeutics orantineoplastic compounds). The other compounds may be administeredconcurrently. As used herein, the word “concurrently” means sufficientlyclose in time to produce a combined effect (that is, concurrently may besimultaneously, or it may be two or more administrations occurringbefore or after each other) As used herein, the phrase “radiationtherapy” includes, but is not limited to, x-rays or gamma rays which aredelivered from either an externally applied source such as a beam or byimplantation of small radioactive sources. Examples of other suitablechemotherapeutic agents which may be concurrently administered withactive agents as described herein include, but are not limited to,Alkylating agents (including, without limitation, nitrogen mustards,ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes):Uracil mustard, Chlormethine, Cyclophosphamide (Cytoxan®), Ifosfamide,Melphalan, Chlorambucil, Pipobroman, Triethylene-melamine,Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,Streptozocin, Dacarbazine, and Temozolomide; Antimetabolites (including,without limitation, folic acid antagonists, pyrimidine analogs, purineanalogs and adenosine deaminase inhibitors): Methotrexate,5-Fluorouracil, Floxuridine, Cytarabine, 6-Mercaptopurine,6-Thioguanine, Fludarabine phosphate, Pentostatine, and Gemcitabine;Natural products and their derivatives (for example, vinca alkaloids,antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxins):Vinblastine, Vincristine, Vindesine, Bleomycin, Dactinomycin,Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Ara-C, paclitaxel(paclitaxel is commercially available as Taxol®), Mithramycin,Deoxyco-formycin, Mitomycin-C, L-Asparaginase, Interferons (especiallyIFN-α), Etoposide, and Teniposide; Other anti-proliferative cytotoxicagents are navelbene, CPT-11, anastrazole, letrazole, capecitabine,reloxafine, cyclophosphamide, ifosamide, and droloxafine. Additionalanti-proliferative cytotoxic agents include, but are not limited to,melphalan, hexamethyl melamine, thiotepa, cytarabin, idatrexate,trimetrexate, dacarbazine, L-asparaginase, camptothecin, topotecan,bicalutamide, flutamide, leuprolide, pyridobenzoindole derivatives,interferons, and interleukins. Preferred classes of antiproliferativecytotoxic agents are the EGFR inhibitors, Her-2 inhibitors, CDKinhibitors, and Herceptin® (trastuzumab). (see, e.g., U.S. Pat. No.6,537,988; U.S. Pat. No. 6,420,377). Such compounds may be given inaccordance with techniques currently known for the administrationthereof.

F. Antibodies

Antibodies and the production thereof are known. See, e.g., U.S. Pat.No. 6,849,719; see also U.S. Pat. Nos. 6,838,282; 6,835,817; 6,824,989.

Antibodies of the invention include antibodies that are modified, i.e.,by the covalent attachment of any type of molecule to the antibody suchthat covalent attachment does not prevent the antibody from specificallybinding to its binding site. For example, antibodies of the inventionmay be modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, or with other protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand or other protein,etc. Any of numerous chemical modifications may be carried out by knowntechniques, including but not limited to specific chemical cleavage,acetylation, formylation, metabolic synthesis of tunicamycin, etc.Additionally, the antibodies may contain one or more non-classical aminoacids.

Polyclonal antibodies of the invention can be generated by any suitablemethod known in the art. For example, a suitable antigen can beadministered to various host animals including, but not limited to,rabbits, mice, rats, etc. to induce the production of sera containingpolyclonal antibodies specific for the antigen. Various adjuvants may beused to increase the immunological response, depending on the hostspecies, and include but are not limited to, Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and corynebacterium parvum.

Monoclonal antibodies can be prepared using a wide variety of techniquesincluding the use of hybridoma, recombinant, and phage displaytechnologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thosetaught, for example, in Harlow et al., Antibodies: A Laboratory Manual,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al.,in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981). The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and known. Briefly, mice are immunizedwith an antigen or a cell expressing such antigen. Once an immuneresponse is detected, e.g., antibodies specific for the antigen aredetected in the mouse serum, the mouse spleen is harvested andsplenocytes isolated. The splenocytes are then fused by known techniquesto any suitable myeloma cells, for example cells from cell line SP20available from the ATCC. Hybridomas are selected and cloned by limiteddilution. The hybridoma clones are then assayed by methods known in theart for cells that secrete antibodies capable of binding a polypeptideof the invention. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)₂ fragments). F(ab′)₂ fragments contain thevariable region, the light chain constant region and the CHI domain ofthe heavy chain.

For example, antibodies can also be generated using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of phage particles whichcarry the polynucleotide sequences encoding them. In a particular, suchphage can be utilized to display antigen-binding domains expressed froma repertoire or combinatorial antibody library (e.g., human or murine).Phage expressing an antigen binding domain that binds the antigen ofinterest can be selected or identified with antigen, e.g., using labeledantigen or antigen bound or captured to a solid surface or bead. Phageused in these methods are typically filamentous phage including fd andM13 binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention include butare not limited to those disclosed in U.S. Pat. Nos. 5,698,426;5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and5,969,108.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art.

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988).

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporatedherein by reference in their entireties. Humanized antibodies areantibody molecules from non-human species antibody that binds thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and framework regions from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (see, e.g., U.S. Pat. Nos. 5,225,539; 5,530,101;and 5,585,089), veneering or resurfacing (see, e.g., EP 592,106; EP519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnickaet al., Protein Engineering 7(6):805-814 (1994); Roguska et al., PNAS91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).

Completely human antibodies are desirable for therapeutic treatment,diagnosis, and/or detection of human patients. Human antibodies can bemade by a variety of methods known in the art including phage displaymethods described above using antibody libraries derived from humanimmunoglobulin sequences. See, e.g., U.S. Pat. Nos. 4,444,887 and4,716,111.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825;5,661,016; 5,545,806; 5,814,318; and 5,939,598.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For exampleantibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention as described above. Thepolynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligation of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR. Alternatively, apolynucleotide encoding an antibody may be generated from nucleic acidfrom a suitable source. If a clone containing a nucleic acid encoding aparticular antibody is not available, but the sequence of the antibodymolecule is known, a nucleic acid encoding the immunoglobulin may beobtained from a suitable source (e.g., an antibody cDNA library, or acDNA library generated from, or nucleic acid, preferably poly A+RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody of the invention) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR may then be cloned into replicable cloning vectorsusing any method well known in the art.

G. Implants

Active compounds of the invention, particularly antagonists such asantibodies or peptides as described above, may be coupled to orconjugated to implants or implantable medical devices in accordance withknown techniques for carrying out the methods described herein, or forcombating problems associated with the implant such as stenosis andrestenosis. See, e.g., U.S. Pat. Nos. 6,786,922; 6,746,686; 6,718,208;6,617,142; 6,352,832; 6,238,872. Any implant can be so utilized,including but not limited to stents (e.g., vascular stents), electrodes,catheters, leads, implantable pacemaker or cardioverter housings,joints, screws, rods, ophthalmic implants (including, but not limitedto, intraocular lens implants, glaucoma implants or drainage implants,and punctal implants or plugs), etc. The implants may be of any suitablematerial, including but not limited to organic polymers (includingstable or inert polymers and biodegradable polymers), metals such asstainless steel and titanium, inorganic materials such as silicon, andcomposites thereof.

H. Screening Assays

The methods, storage media, data structures, and the like, along withcompounds identified by such methods and methods of use thereof, may beimplemented accordance with known techniques such as described in L.Tong et al., PCT Application WO 2004/063715, titled Methods of UsingCrystal Structure of Carboxyltransferase Domain of Acetyl-CoACarboxylase, Modulators Thereof and Computer Methods.

Advantageously the crystal structure of β3 integrin, including thecysteine loop domain thereof, is known, although not known for thepurposes described herein.

Peterson et al., Biochemistry 44:565, 2005 have utilized small anglelight scattering, and in an earlier publication, NMR, to determine tothe three dimensional structure of vitronectin. In their model theheparin binding domain is surface exposed and clearly distinct from theRGD binding domain as well as the plasminogen activator inhibitor-1binding domain. Although the heparin binding domain is not cryptic theycomment that polymerized vitronectin is likely to bind more avidly toheparin due to an even better exposure of this domain. Since multimericforms of vitronectin occur in disease states such as atherosclerosis andin vitronectin that is associated with extracellular matrix, this mayhave pathophysiologic significance. Similarly the crystal structure ofthe extracellular portion of αVβ3 dimer with and without being complexedto RGD ligands has been reported (Science 294:339, 2001 and Science296:151, 2002). These two papers report the molecular coordinates of thecysteine loop structure between residues cysteine 177 and cysteine 184hereafter termed the cysteine loop. This structure is mapped and thethree dimensional structure is illustrated in FIG. 6 of the firstmanuscript. The paper clearly illustrates that this binding site isdistinct from the RGD sequence binding site. It is described as a ligandspecificity region but no further description of its functionalproperties are given in this publication. It is clear from the crystalstructure that its surface is exposed. Similarly the active confirmationof the protein results in further surface exposure of this bindingdomain. The molecular coordinates are described and the GI extension #'sare 232004340 20664279 and 16975254. Similarly the extension numbers ofthe crystal structure are IL5GAZ-A5 and B0, B6, 7, 8, 9, 10 and C0.

The Protein Data Bank (PDB) accession numbers for the unliganded and RGDliganded structures of αVβ3 integrin are 1JV2 and 1L5G, the disclosuresof which are incorporated herein by reference in their entirety fortheir teachings. Tables 1 and 2 presented below provide the molecularcoordinates for amino acid residues between cysteine 177 and cysteine184 of the unliganded and RGD liganded structures of β3 integrin,respectively.

TABLE 1 Atomic coordinates for amino acid residues betweencysteine 177 and cysteine 184 of β3 integrin CRYST1130.000   130.000   307.300   90.00   90.00   120.00 P 32 2 1           6ORIGX1     1.000000 0.000000 0.000000 0.00000 ORIGX2     0.0000001.000000 0.000000 0.00000 ORIGX3     0.000000 0.000000 1.000000 0.00000SCALE1     0.007692 0.004441 0.000000 0.00000 SCALE2     0.0000000.008882 0.000000 0.00000 SCALE3     0.000000 0.000000 0.003254 0.00000ATOM 8193 N CYS B 177 30.617 38.285 38.020 1.00 51.69 N ATOM 8194 CACYS B 177 29.881 38.955 39.076 1.00 61.73 C ATOM 8195 C CYS B 177 30.99739.840 39.641 1.00 63.92 C ATOM 8196 O CYS B 177 31.309 39.784 40.8191.00 67.08 O ATOM 8197 CB CYS B 177 29.473 37.927 40.157 1.00 67.19 CATOM 8198 SG CYS B 177 28.097 36.748 39.847 1.00 89.98 S ATOM 8199 NTYR B 178 31.638 40.596 38.756 1.00 64.48 N ATOM 8200 CA TYR B 17832.764 41.495 39.061 1.00 64.75 C ATOM 8201 C TYR B 178 32.786 42.37040.337 1.00 64.07 C ATOM 8202 O TYR B 178 33.761 42.313 41.085 1.0062.11 O ATOM 8203 CB TYR B 178 33.022 42.386 37.846 1.00 74.79 C ATOM8204 CG TYR B 178 31.983 43.478 37.675 1.00 89.90 C ATOM 8205 CD1TYR B 178 30.617 43.197 37.784 1.00 100.00 C ATOM 8206 CD2 TYR B 17832.364 44.805 37.472 1.00 97.33 C ATOM 8207 CE1 TYR B 178 29.656 44.21437.704 1.00 100.00 C ATOM 8208 CE2 TYR B 178 31.409 45.833 37.391 1.00100.00 C ATOM 8209 CZ TYR B 178 30.058 45.528 37.508 1.00 100.00 C ATOM8210 OH TYR B 178 29.105 46.526 37.450 1.00 99.70 O ATOM 8211 NASP B 179 31.761 43.212 40.540 1.00 61.63 N ATOM 8212 CA ASP B 17931.664 44.120 41.705 1.00 62.19 C ATOM 8213 C ASP B 179 32.085 43.41342.972 1.00 68.31 C ATOM 8214 O ASP B 179 33.148 43.669 43.539 1.0070.06 O ATOM 8215 CB ASP B 179 30.224 44.597 41.919 1.00 60.12 C ATOM8216 CG ASP B 179 29.704 45.424 40.782 1.00 66.56 C ATOM 8217 OD1ASP B 179 28.590 45.129 40.294 1.00 60.98 O ATOM 8218 OD2 ASP B 17930.399 46.381 40.386 1.00 80.12 O ATOM 8219 N MET B 180 31.209 42.51943.415 1.00 79.09 N ATOM 8220 CA MET B 180 31.470 41.708 44.591 1.0088.86 C ATOM 8221 C MET B 180 32.586 40.865 43.984 1.00 90.63 C ATOM8222 O MET B 180 32.520 40.553 42.794 1.00 92.27 O ATOM 8223 CBMET B 180 30.219 40.863 44.929 1.00 98.30 C ATOM 8224 CG MET B 18029.616 40.019 43.759 1.00 100.00 C ATOM 8225 SD MET B 180 27.803 40.09943.408 1.00 91.76 S ATOM 8226 CE MET B 180 27.856 41.139 41.950 1.0088.30 C ATOM 8227 N LYS B 181 33.663 40.604 44.724 1.00 89.72 N ATOM8228 CA LYS B 181 34.763 39.829 44.143 1.00 93.91 C ATOM 8229 CLYS B 181 34.430 38.380 43.748 1.00 93.74 C ATOM 8230 O LYS B 181 35.07437.810 42.857 1.00 94.02 O ATOM 8231 CB LYS B 181 36.004 39.857 45.0471.00 98.76 C ATOM 8232 CG LYS B 181 37.208 39.133 44.442 1.00 100.00 CATOM 8233 CD LYS B 181 38.370 39.089 45.410 1.00 100.00 C ATOM 8234 CELYS B 181 39.471 38.167 44.899 1.00 100.00 C ATOM 8235 NZ LYS B 18140.690 38.200 45.762 1.00 100.00 N ATOM 8236 N THR B 182 33.382 37.83644.360 1.00 92.50 N ATOM 8237 CA THR B 182 32.941 36.458 44.122 1.0095.53 C ATOM 8238 C THR B 182 32.590 36.101 42.662 1.00 94.42 C ATOM8239 O THR B 182 31.417 36.137 42.272 1.00 100.00 O ATOM 8240 CBTHR B 182 31.723 36.138 45.009 1.00 99.31 C ATOM 8241 OG1 THR B 18231.913 36.732 46.313 1.00 100.00 O ATOM 8242 CG2 THR B 182 31.526 34.63245.150 1.00 99.19 C ATOM 8243 N THR B 183 33.584 35.681 41.879 1.0092.69 N ATOM 8244 CA THR B 183 33.346 35.309 40.483 1.00 88.83 C ATOM8245 C THR B 183 32.313 34.189 40.349 1.00 83.16 C ATOM 8246 O THR B 18332.163 33.327 41.224 1.00 81.19 O ATOM 8247 CB THR B 183 34.653 34.91539.734 1.00 87.66 C ATOM 8248 OG1 THR B 183 35.311 33.836 40.416 1.0093.17 O ATOM 8249 CG2 THR B 183 35.597 36.121 39.631 1.00 90.15 C ATOM8250 N CYS B 184 31.593 34.226 39.236 1.00 78.27 N ATOM 8251 CACYS B 184 30.543 33.265 38.955 1.00 71.84 C ATOM 8252 C CYS B 184 30.52832.977 37.459 1.00 68.43 C ATOM 8253 O CYS B 184 31.391 33.445 36.7191.00 68.40 O ATOM 8254 CB CYS B 184 29.193 33.867 39.372 1.00 78.22 CATOM 8255 SG CYS B 184 28.756 35.396 38.458 1.00 81.45 S

TABLE 2Atomic coordinates for amino acid residues between cysteine 177 andcysteine 184 of β3 integrin complexed with an Arg-Gly-Asp (RGD) ligandCRYST1129.790  129.790  308.780  90.00  90.00  120.00 P 32 2 1           6ORIGX1 1.000000 0.000000 0.000000 0.00000 ORIGX2 0.000000 1.0000000.000000 0.00000 ORIGX3 0.000000 0.000000 1.000000 0.00000 SCALE10.007705 0.004448 0.000000 0.00000 SCALE2 0.000000 0.008897 0.0000000.00000 SCALE3 0.000000 0.000000 0.003239 0.00000 ATOM 8193 N CYS B 17729.514 39.700 37.901 1.00 39.17 N ATOM 8194 CA CYS B 177 29.723 40.09139.299 1.00 49.86 C ATOM 8195 C CYS B 177 31.188 40.306 39.619 1.0051.72 C ATOM 8196 O CYS B 177 31.741 39.770 40.581 1.00 52.27 O ATOM8197 CB CYS B 177 29.045 39.124 40.271 1.00 48.58 C ATOM 8198 SGCYS B 177 29.786 37.472 40.399 1.00 47.78 S ATOM 8199 N TYR B 178 31.80641.091 38.742 1.00 55.89 N ATOM 8200 CA TYR B 178 33.192 41.474 38.8351.00 56.91 C ATOM 8201 C TYR B 178 33.247 42.935 39.269 1.00 55.86 CATOM 8202 O TYR B 178 34.278 43.428 39.698 1.00 55.78 O ATOM 8203 CBTYR B 178 33.886 41.150 37.519 1.00 60.90 C ATOM 8204 CG TYR B 17834.649 42.246 36.813 1.00 70.75 C ATOM 8205 CD1 TYR B 178 34.050 43.46436.482 1.00 72.46 C ATOM 8206 CD2 TYR B 178 35.952 42.013 36.363 1.0082.19 C ATOM 8207 CE1 TYR B 178 34.732 44.415 35.712 1.00 85.76 C ATOM8208 CE2 TYR B 178 36.638 42.943 35.596 1.00 86.77 C ATOM 8209 CZTYR B 178 36.026 44.137 35.270 1.00 89.20 C ATOM 8210 OH TYR B 17836.707 45.026 34.463 1.00 91.89 O ATOM 8211 N ASP B 179 32.110 43.61639.165 1.00 58.12 N ATOM 8212 CA ASP B 179 31.996 44.992 39.625 1.0062.70 C ATOM 8213 C ASP B179 31.630 44.883 41.112 1.00 64.92 C ATOM 8214O ASP B179 30.827 45.659 41.645 1.00 62.59 O ATOM 8215 CB ASP B17930.929 45.780 38.833 1.00 63.77 C ATOM 8216 CG ASP B179 29.536 45.15538.899 1.00 67.28 C ATOM 8217 OD1 ASP B179 29.164 44.447 37.938 1.0073.03 O ATOM 8218 OD2 ASP B179 28.800 45.397 39.886 1.00 59.16 O ATOM8219 N MET B180 32.200 43.857 41.747 1.00 66.78 N ATOM 8220 CA MET B18032.010 43.546 43.162 1.00 74.44 C ATOM 8221 C MET B180 33.018 42.46343.546 1.00 75.25 C ATOM 8222 O MET B180 33.614 42.506 44.625 1.00 79.74O ATOM 8223 CB MET B180 30.585 43.043 43.437 1.00 74.85 C ATOM 8224 CGMET B180 30.158 41.826 42.616 1.00 72.71 C ATOM 8225 SD MET B180 28.77640.893 43.337 1.00 69.56 S ATOM 8226 CE MET B180 27.571 42.205 43.6191.00 71.52 C ATOM 8227 N LYS B181 33.189 41.504 42.633 1.00 76.92 N ATOM8228 CA LYS B181 34.102 40.354 42.748 1.00 77.50 C ATOM 8229 C LYS B18133.709 39.072 43.481 1.00 72.04 C ATOM 8230 O LYS B181 33.668 39.01244.714 1.00 72.51 O ATOM 8231 CB LYS B181 35.525 40.773 43.147 1.0079.56 C ATOM 8232 CG LYS B181 36.399 41.191 41.967 1.00 80.77 C ATOM8233 CD LYS B181 35.869 40.672 40.615 1.00 81.67 C ATOM 8234 CE LYS B18135.834 39.138 40.467 1.00 85.27 C ATOM 8235 NZ LYS B181 34.951 38.70439.323 1.00 80.43 N ATOM 8236 N THR B182 33.503 38.032 42.678 1.00 63.31N ATOM 8237 CA THR B182 33.138 36.694 43.133 1.00 63.32 C ATOM 8238 CTHR B182 33.327 35.778 41.921 1.00 62.57 C ATOM 8239 O THR B182 33.16734.561 42.003 1.00 67.53 O ATOM 8240 CB THR B182 31.670 36.635 43.6501.00 64.03 C ATOM 8241 OG1 THR B182 31.565 37.379 44.873 1.00 70.53 OATOM 8242 CG2 THR B182 31.225 35.189 43.915 1.00 61.10 C ATOM 8243 NTHR B183 33.718 36.385 40.805 1.00 59.72 N ATOM 8244 CA THR B183 33.96035.682 39.549 1.00 60.67 C ATOM 8245 C THR B183 32.872 34.710 39.0651.00 56.82 C ATOM 8246 O THR B183 33.177 33.743 38.357 1.00 59.75 O ATOM8247 CB THR B183 35.333 34.960 39.557 1.00 58.54 C ATOM 8248 OG1THR B183 35.680 34.597 40.895 1.00 53.98 O ATOM 8249 CG2 THR B183 36.41935.845 38.966 1.00 61.78 C ATOM 8250 N CYS B184 31.612 34.971 39.4241.00 52.33 N ATOM 8251 CA CYS B184 30.493 34.121 38.998 1.00 43.94 CATOM 8252 C CYS B184 30.506 34.081 37.486 1.00 38.56 C ATOM 8253 OCYS B184 30.486 35.116 36.837 1.00 35.49 O ATOM 8254 CB CYS B184 29.16134.704 39.457 1.00 46.67 C ATOM 8255 SG CYS B184 28.929 36.411 38.8951.00 44.19 S

In general any method known to those skilled in the art may be used toprocess X-ray diffraction data. In addition, in order to determine theatomic structure of a β3 integrin according to the present invention,multiple isomorphous replacement (MIR) analysis, model building andrefinement may be performed. For MIR analysis, the crystals may besoaked in heavy-atoms to produce heavy atom derivatives necessary forMIR analysis. As used herein, heavy atom derivative or derivatizationrefers to the method of producing a chemically modified form of aprotein or protein complex crystal wherein said protein is specificallybound to a heavy atom within the crystal. In practice a crystal issoaked in a solution containing heavy metal atoms or salts, ororganometallic compounds, e.g., lead chloride, gold cyanide, thimerosal,lead acetate, uranyl acetate, mercury chloride, gold chloride, etc.,which can diffuse through the crystal and bind specifically to theprotein. The location(s) of the bound heavy metal atom(s) or salts canbe determined by X-ray diffraction analysis of the soaked crystal. Thisinformation is used to generate MIR phase information which is used toconstruct the three-dimensional structure of the crystallized cysteineloop domain of a β3 integrin. Thereafter, an initial model of thethree-dimensional structure may be built using the program O (Jones etal., 1991, Acta Crystallogr. A47:110-119). The interpretation andbuilding of the structure may be further facilitated by use of theprogram CNS (Brunger et al., 1998, Acta Crystallogr. D54:905-921).

The method of molecular replacement broadly refers to a method thatinvolves generating a preliminary model of the three-dimensionalstructure of crystal of a cysteine loop structure of a β3 integrin ofthe present invention. Molecular replacement is achieved by orientingand positioning a molecule whose structural coordinates are known withinthe unit cell as defined by the X-ray diffraction pattern obtained fromthe cysteine loop domain of a β3 integrin under study (or thecorresponding enzyme/substrate complex or enzyme/inhibitor complex) soas to best account for the observed diffraction pattern of the unknowncrystal. Phases can then be calculated from this model and combined withthe observed amplitudes to give an approximate Fourier synthesis of thestructure. This in turn can be subject to any of several forms ofrefinement to provide a final, accurate structure. The molecularreplacement method may be applied using techniques known to the skilledartisan.

The three-dimensional structures and the specific atomic coordinatesassociated with said structures of the cysteine loop domain of β3, aloneor in complex with a substrate or specific binding ligand such asheparin, are useful for solving the structure of crystallized forms ofheparin binding domains of other β3 ligands. Such proteins comprise aroot mean square deviation (RMSD) of no greater than 2.0 Å, 1.5 Å, 1.0 Åor 0.5 Å in the positions of Cα atoms for at least 50 percent or more ofthe amino acids of the structure of the cysteine loop domain of the β3integrin. Such an RMSD may be expected based on the amino acid sequenceidentity. Chothia and Lesk, 1986, EMBO J. 5:823-826.

Modulators of a β3 integrin, and hence of IGF-1 activity, can bedesigned using three-dimensional structures obtained as set forth in thepreceding section and the Examples section below. These structures maybe used to design or screen for molecules that are able to form thedesired interactions with one or more binding sites of the cysteine loopdomain.

The models of the cysteine loop domain (and sub-regions, includingactive sites, binding sites or cavities thereof) of a β3 integrindescribed herein may be used to either directly develop a modulator fora β3 integrin. The ability for such a modulator to modulate the activityof a cysteine loop domain of a β3 integrin can be confirmed by furthercomputer analysis, and/or by in vitro and/or in vivo testing.

A model of a cysteine loop domain may be comprised in a virtual oractual protein structure that is smaller than, larger than, or the samesize as a native cysteine loop domain of a β3 integrin protein. Theprotein environment surrounding the active site model may be homologousor identical to the native cysteine loop domain of a β3 integrin, or itmay be partially or completely non-homologous.

Thus, the present invention provides for a method for rationallydesigning a modulator of a β3 integrin, comprising the steps of (i)producing a computer readable model of a molecule comprising a region(i.e., an active site, reactive site, or a binding site) of a cysteineloop domain of β3 integrin (e.g. human or pig β3 integrin); and (ii)using the model to design a test compound having a structure and acharge distribution compatible with (i.e. able to be accommodatedwithin) the region of the cysteine loop domain, wherein the testcompound can comprise a functional group that may interact with theactive site to modulate activity. If the crystal structure is notavailable for the cysteine loop domain to be examined, homology modelingmethods known to those of ordinary skill in the art may be used toproduce a model, which then may be used to design test compounds asdescribed above.

The atomic coordinates of atoms of the cysteine loop domain (or aregion/portion thereof) of a β3 integrin or a β3 integrin-related enzymemay be used in conjunction with computer modeling using a dockingprogram such as GRAM, DOCK, HOOK or AUTODOCK (Dunbrack et al., 1997,Folding & Design 2:27-42) to identify potential modulators. Thisprocedure can include computer fitting of potential modulators to amodel of a cysteine loop domain (including models of regions of acysteine loop domain, for example, an active site, or a binding site) toascertain how well the shape and the chemical structure of the potentialmodulator will complement the active site or to compare the potentialmodulators with the binding of substrate or known inhibitor molecules inthe active site.

Computer programs may be employed to estimate the attraction, repulsionand/or steric hindrance associated with a postulated interaction betweenthe reactive site model and the potential modulator compound. Generally,characteristics of an interaction that are associated with modulatoractivity include, but are not limited to, tight fit, low sterichindrance, positive attractive forces, and specificity.

Modulator compounds of the present invention may also be designed byvisually inspecting the three-dimensional structure of a reactive siteof the cysteine loop domain of a β3 integrin, a technique known in theart as “manual” drug design. Manual drug design may employ visualinspection and analysis using a graphics visualization program known inthe art.

As an alternative or an adjunct to rationally designing modulators,random screening of a small molecule library, a peptide library or aphage library for compounds that interact with and/or bind to asite/region of interest (i.e., a binding site, active site or a reactivesite, for example) of the cysteine loop domain of a β3 integrin may beused to identify useful compounds. Such screening may be virtual; smallmolecule databases can be computationally screened for chemical entitiesor compounds that can bind to or otherwise interact with a virtual modelof an active site, binding site or reactive site of a cysteine loopdomain of a β3 integrin. Alternatively, screening can be against actualmolecular models of the cysteine loop domain or portions thereof.Further, antibodies can be generated that bind to a site of interest ofthe cysteine loop domain of β3. After candidate (or “test”) compoundsthat can bind to the cysteine loop domain are identified, the compoundscan then be tested to determine whether they can modulate cysteine loopdomain activity (see below).

In one embodiment, β3 integrins containing cysteine loop domains,nucleic acids, and cells containing and/or expressing the cysteine loopdomains are used in screening assays. Screens may be designed to firstfind candidate compounds that can bind to a cysteine loop domain orportion thereof, and then these compounds may be used in assays thatevaluate the ability of the candidate compound to modulate the cysteineloop domain or β3 integrin activity. Thus, as will be appreciated bythose in the art, there are a number of different assays which may berun, including binding assays and activity assays. In one aspect,candidate compounds are first tested to determine whether they can bindto a particular binding site of the heparin binding domain.

Thus, in one embodiment, the methods comprise combining a cysteine loopdomain or portion thereof and a candidate compound, and determining thebinding of the candidate compound to the cysteine loop domain or portionthereof. In some embodiments of the methods herein, the cysteine loopdomain (or portion thereof) or the candidate agent is non-diffusablybound to an insoluble support having isolated sample receiving areas(e.g., a microtiter plate, an array, etc.). The insoluble supports maybe made of any composition to which the compositions can be bound, isreadily separated from soluble material, and is otherwise compatiblewith the overall method of screening. The surface of such supports maybe solid or porous and of any convenient shape. Examples of suitableinsoluble supports include microtiter plates, arrays, membranes andbeads. These are typically made of glass, plastic (e.g., polystyrene),polysaccharides, nylon or nitrocellulose, Teflon™, etc. Microtiterplates and arrays are especially convenient because a large number ofassays can be carried out simultaneously, using small amounts ofreagents and samples—i.e., they enable high-throughput screening.Following binding of the cysteine loop domain, excess unbound materialis removed by washing. The sample receiving areas may then be blockedthrough incubation with bovine serum albumin (BSA), caseine or otherinnocuous protein or other moiety.

A candidate compound is added to the assay. Candidate compounds include,but are not limited to, specific antibodies, compounds from chemicallibraries, peptide analogs, etc. Of particular interest are screeningassays for compounds that have a low toxicity for human cells. A widevariety of assays may be used for this purpose, including labeled invitro protein-protein binding assays, immunoassays for protein binding,NMR assays to determine protein-protein or protein-chemical compoundbinding, and the like. Candidate compounds can also includeinsecticides, herbicides or fungicides.

The term “candidate compound” as used herein describes any molecule,e.g., protein, oligopeptide, small organic molecule, polysaccharide,polynucleotide, etc., with the capability of directly or indirectlymodulating heparin binding domain or β3 integrin activity. Generally aplurality of assay mixtures are run in parallel with different compoundconcentrations to obtain a differential response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, i.e., at zero concentration or below the level ofdetection.

Candidate compounds can encompass numerous chemical classes, thoughtypically they are organic molecules, and in one embodiment they aresmall organic compounds having a molecular weight of more than 100 andless than about 2,500 daltons. Candidate compounds can comprisefunctional groups necessary for structural interaction with proteins,for example hydrogen bonding, and typically include at least an amine,carbonyl, hydroxyl or carboxyl group, preferably at least two of thefunctional chemical groups. The candidate compounds can comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.

Candidate compounds can be obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including combinatorialchemical synthesis and the expression of randomized peptides oroligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs. In one embodiment, the library is fully randomized,with no sequence preferences or constants at any position. In another,the library is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities.

The determination of the binding of the candidate compound to thecysteine loop domain may be done in a number of ways. In one embodiment,the candidate compound is labeled, and binding determined directly. Forexample, this may be done by attaching all or a portion of the cysteineloop domain to a solid support, adding a labeled candidate compound (forexample a fluorescent label or radioactive label), washing off excessreagent, and determining whether the label is present on the solidsupport. Various blocking and washing steps may be utilized as is knownin the art.

By “labeled” herein is meant that the compound is either directly orindirectly labeled with a label which provides a detectable signal,e.g., radioisotope, fluorescers, enzyme, antibodies, particles such asmagnetic particles, chemiluminescers, or specific binding molecules,etc. Specific binding molecules include pairs, such as biotin andstreptavidin, digoxin and antidigoxin, etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined above. The label can directly or indirectlyprovide a detectable signal.

In one embodiment, the binding of the candidate compound is determinedthrough the use of competitive binding assays. In this embodiment, thecompetitor is a binding moiety known to bind to the cysteine loopdomain, such as an antibody, peptide, ligand, etc. Under certaincircumstances, there may be competitive binding as between the candidatecompound and the known binding moiety, with the binding moietydisplacing the bioactive agent.

In one embodiment, the candidate compound is labeled. Either thecandidate compound, or the competitor, or both, is added first to thecysteine loop domain for a time sufficient to allow binding, if present.Incubations may be performed at any temperature which facilitatesoptimal binding, typically between 4 and 40° C. Incubation periods areselected for optimum binding but may also be optimized to facilitaterapid high-throughput screening. Typically between 0.1 and 1 hour willbe sufficient. Excess reagent is generally removed or washed away. Thesecond component is then added, and the presence or absence of thelabeled component is followed, to indicate binding.

In one embodiment, the competitor is added first, followed by thecandidate compound. Displacement of the competitor is an indication thatthe candidate compound is binding to the cysteine loop domain and thusis capable of binding to, and potentially modulating, the activity ofthe cysteine loop domain. In this embodiment, either component can belabeled. Thus, for example, if the competitor is labeled, the presenceof label in the wash solution indicates displacement of the competitorby the candidate compound. Alternatively, if the candidate compound islabeled, the presence of the label on the support indicates displacementof the candidate compound.

In one embodiment, a potential ligand for a cysteine loop domain can beobtained by screening a recombinant bacteriophage library (Scott andSmith, Science, 249:386-390 (1990); Cwirla et al., Proc. Natl. Acad.Sci., 87:6378-6382 (1990); Devlin et al., Science, 249:404-406 (1990).Specifically, the phage library can be mixed in low dilutions withpermissive E. coli in low melting point LB agar which is then poured ontop of LB agar plates. After incubating the plates at 37° C. for aperiod of time, small clear plaques in a lawn of E. coli will form whichrepresents active phage growth and lysis of the E. coli. Arepresentative of these phages can be absorbed to nylon filters byplacing dry filters onto the agar plates. The filters can be marked fororientation, removed, and placed in washing solutions to block anyremaining absorbent sites. The filters can then be placed in a solutioncontaining, for example, a radioactive heparin binding domain (orportion thereof). After a specified incubation period, the filters canbe thoroughly washed and developed for autoradiography. Plaquescontaining the phage that bind to the radioactive heparin binding domainor portion thereof can then be identified. These phages can be furthercloned and then retested for their ability to bind to the cysteine loopdomain as before. Once the phages have been purified, the bindingsequence contained within the phage can be determined by standard DNAsequencing techniques. Once the DNA sequence is known, syntheticpeptides can be generated which represents these sequences, and furtherbinding studies can be performed as discussed herein.

In another embodiment, a potential ligand for a cysteine loop domain canbe obtained by screening candidate compounds by NMR (see for example,U.S. Patent Application Publication No. US2003/0148297A1 or Pellecchiaet al., Nature Reviews Drug Discovery, 1:211-219 (2002)). As mentioned,a cysteine loop domain or portions thereof can be immobilized to alltypes of solid supports. It is not needed that the binding be a covalentbinding. It is only required that the target is kept immobilized in theNMR measuring environment. Moreover, the immobilization need not bedirectly to the solid support; it may also occur indirectly throughsuitable bridging moieties or molecules, or through spacers. Verysuitable supports are solid polymers used in chromatography, such aspolystyrene, sepharose and agarose resins and gels, e.g. in bead form orin a porous matrix form. Additionally, appropriately chemically modifiedsilicon based materials are also very suitable supports.

Any soluble molecule can be used as a compound that is a candidate tobinding to the cysteine loop domain. It is not necessary that the saidsoluble molecule is water-soluble. Any liquid medium that does notdenature the said compound nor the cysteine loop domain molecule can beused in the NMR measurements. The cysteine loop domain target moleculeis immobilized to a suitable support, such as a solid resin, andadditionally placed in a suitable NMR probe, for example, a flowinjection NMR probe, for the duration of the screening. Each sample ofthe compounds to be screened, e.g. the compounds from a library, is thenapplied to the immobilized target by pumping it through, along or viathe solid support. The sample to be assayed may contain a singlecomponent suspected of binding to the cysteine loop domain targetmolecule, or may contain multiple components of a compound library orother type of collection or mixture. The flow may be stopped when adesired level of concentration of the compounds to be assayed is reachedin the target containing probe or vessel.

For the acquisition of the NMR spectra, in principle any NMR pulsesequence capable of detecting resonances from dissolved molecule samplesand, preferably suppressing residual solvent signals, such as by pulsedfield gradients, may be used to detect binding. In practice, however, aone-dimensional ¹H-NMR spectrum is acquired with sufficient resolutionand sensitivity to detect and quantitate resonances derived from eachcompound being assayed in the presence of the control solid support. Inaddition, a second spectrum recorded using the same NMR protocol, isacquired for the same solution of screenable compounds in the presenceof the solid support containing the immobilized cysteine loop domaintarget molecule. Optionally, a third spectrum may be acquired in thepresence of the solid support containing the immobilized cysteine loopdomain target molecule in order to detect extremely weak target binding.This spectrum can be recorded while using a diffusion or T2 filter.

After acquisition of the NMR spectrum, the sample of small compound orcompounds is washed out of the NMR probe containing the targetimmobilized solid support. Subsequently, the next sample can be appliedto the probe in a stopped-flow manner. Throughout the entire screeningprocess a single sample of the target immobilized solid support remainsin the NMR probe. The target immobilized solid support need only bechanged should the target become denatured, chemically degraded orsaturated by a tight-binding compound that cannot be washed away. Inorder to safeguard that certain compounds do not bind in such a way thatthe target molecule is blocked, at certain stages, a control is carriedout to check the availability of binding opportunities to the targetmolecule.

The NMR spectra are preferably compared by subtracting one of the twoNMR data sets from the other, thereby creating a difference spectrum. Ingeneral, since the target molecule is essentially in the solid phase,the resonances from compounds that bind to the target molecule arebroadened beyond detection while in the bound state. Thus, binding issensitively and reliably detectable by a decrease in height of peaksthat derive exclusively from the solution form of compounds binding tothe target molecule. This effect is most easily seen in the differencespectra. An alternative approach that can be used to quantitate theaffinity of the target-ligand interaction is to determine peak areas(e.g. by integrating) in the control and experimental spectra andcompare the values of these areas. Although it is possible to carry outthe NMR screening method in batch mode, in the flow-injection set-up,one sample of target may be used to screen an entire library.

Biological or Biochemical Screening Assays.

Compounds can additionally be screened for activity in modulatingcellular activation by IGF-1 in bioassays or chemical assays of thepresent invention. Compounds identified as modulators or potentialmodulators of IGF-1 activity by methods as described above may befurther screened for specific activity as agonists or antagonists in invivo or in vitro assays in accordance with known techniques, and/or asdiscussed further below.

Assay Methodologies

Methods to Assess Biochemical and Biological Activity of Enhancers orInhibitors of αVβ3.

Modification of IGF-I actions. In addition to competitive bindingassays, in order to determine whether compounds that bind to thecysteine loop binding site on αVβ3 influence IGF-I signaling and actionsit has necessitated the utilization of assays that assess thebiochemical and biologic actions that are stimulated when this site isactivated by ligands and how this alters the cellular responses toIGF-I. Inhibitors will obviously inhibit the ability of IGF-I tostimulate these cellular processes whereas stimulators will facilitateits ability to do so. These assays include but are not totally limitedto the following: β3 subunit phosphorylation, β3 binding to SHPS-1, andintegrin associated protein (IAP) as a complex, the association of IAPwith SHPS-1, SHPS-1 phosphorylation and Shc recruitment to SHPS-1, Shcphosphorylation, stimulation of DNA synthesis and cell replication orcell migration. Ligands that bind to β3 through the cysteine loop domainoften induce both conformational changes and β3 phosphorylation.Similarly, stimulation of β3 phosphorylation can induce a conformationalchange in β3 secondarily. β3 phosphorylation is measured by applying thecompound that binds to β3 to smooth muscle and endothelial cells inculture. First compounds are added using concentrations varying from 0.1to 1 μg/ml to confluent smooth muscle or endothelial cell monolayers in10 cm dishes. Following a fixed time period of exposure to the cells(2-4 hrs) the cells are lysed in 900 μl of RIPA buffer (1,2). Thelysates are either analyzed directly by immunoblotting for β3 to measurepolymerization or immunoprecipitated with an anti β3 antibody and thenimmunoblotted for phosphotyrosine. Immunoblotting is analyzed followingseparation of the proteins contained in 30 μl of cell lysate by SDSpolyacrylamide gel electrophoresis (SDS-PAGE). For immunoprecipitationthe primary β3 antibody is added at a 1:300 dilution to 900 μl of lysateand incubated overnight. The immune complexes are precipitated withprotein A sepharose and eluted with Laemmli sample buffer (Maile L A andClemmons D R, Endocrinology 143: 4259-4264 (2002); Maile L A, Clarke JB, Clemmons D R, J Biol Chem, 277:8955-8960 (2002)). The amount ofphosphorylated β3 is then determined by SDS-PAGE followed byimmunoblotting with a monoclonal anti-phosphotyrosine antibody (PY99)(Ling Y, Maile L A, Clemmons D R, Mol Endocrinol, 17:1824-1833 (2003)).

The methodology for determining complex formation between SHPS-1 and IAPhas been previously published (Maile L A, Clarke J B, Clemmons D R, MolBiol Cell, 14:3519-28 (2003)). Briefly the cells are exposed to testagents that activate β3 through the cysteine loop domain and then theyare exposed to IGF-I. Following IGF-I exposure if β3 is ligand occupiedby an activating ligand IAP and SHPS-1 will associate in a largemolecular weight complex. Importantly if this is completely inhibited byantibody that binds to the cysteine loop domain or other inhibitors,they will not associate. To detect this complex cell lysates areprepared as described previously and immunoprecipitated for SHPS-1 usinga 1:330 dilution of a polyclonal antiserum. The immunoprecipitatedproteins are separated by SDS-PAGE and immunoblotted using a purifiedmonoclonal antibody to detect IAP (B6H12) (Id.).

SHPS-1 Phosphorylation.

To determine SHPS-1 phosphorylation the β3 ligand (either agonist orantagonist) is added to the cultures for periods between 30 minutes and2 hrs at 37° C. IGF-I is then added and cell lysates are prepared atspecific time points. In addition to baseline 3, 5, 10, and 20 minlysates are prepared after exposure to IGF-I. The lysates are preparedas described previously and immunoprecipitated for SHPS-1 usinganti-SHPS-1 polyclonal antiserum at a 1:330 dilution. Theimmunoprecipitate which is pelleted with protein A sepharose is thenanalyzed by SDS-PAGE followed by immunoblotting for phosphotyrosineusing the PY99 monoclonal antibody that detects phosphorylated tyrosineresidues (Maile L A and Clemmons D R, Endocrinology 143: 4259-4264(2002); Maile L A, Clarke J B, Clemmons D R, J Biol Chem, 277:8955-8960(2002); Maile L A and Clemmons D R Circ Res, 93: 925-931 (2003)). Theexpected response is that IGF-I stimulates SHPS-1 phosphorylation andthat agonists will increase either the intensity of SHPS-1phosphorylation or prolong its duration. In contrast, antagonists willdecrease the intensity and shorten its duration.

Shc Phosphorylation.

The binding and recruitment of Shc to SHPS-1 is critical for Shcphosphorylation which is necessary for IGF-I signaling particularly insmooth muscle cells and endothelium in diabetes. To measure Shcphosphorylation cell cultures are exposed to agonists or antagonists asdescribed previously and then cell cultures are then exposed to IGF-Ifor periods of 10, 20 or 30 minutes. Cell lysates are prepared at eachtime point as described previously and immunoprecipitated using a 1:1000dilution of anti-Shc polyclonal antiserum. The immunoprecipitate iscleared with protein A sepharose and then the proteins eluted withLaemmli sample buffer and analyzed by SDS-PAGE followed byimmunoblotting with the anti phosphotyrosine antibody PY99. The expectedresponse is that IGF-I will stimulate Shc phosphorylation. This will besignificantly prolonged and intensified particularly at the later timepoints in cultures exposed to β3 agonists. In contrast, antagonists willinhibit Shc phosphorylation.

Shc Recruitment to SHPS-1.

Cultures are exposed to either agonists or antagonists for the timeperiods listed previously. Cultures are then washed and IGF-I is addedfor periods of 5, 10, 20 or 30 min. Cell lysates are prepared asdescribed previously (Maile L A and Clemmons D R, Endocrinology 143:4259-4264 (2002)) and immunoprecipitated using anti-SHPS-1 antiserausing a 1:330 dilution. Following clearing of the immune complexes withprotein A sepharose, the immunoprecipitates are analyzed by SDS-PAGEfollowed by immunoblotting for Shc using a 1:2000 dilution of anti-Shcantiserum. The expected response is that IGF-I will stimulate Shcrecruitment to SHPS-1 which is required for Shc to undergophosphorylation. However if an antagonist is used, then SHPS-1 will notbe phosphorylated and Shc will not bind to SHPS-1 therefore recruitmentwill be undetectable or greatly diminished.

Activation of MAP kinase. Activation of MAP kinase is critical forstimulation of cell division and cell migration in smooth muscle cellsand endothelium by IGF-I. Shc phosphorylation and recruitment to themembrane as noted previously is required for MAP kinase activation. Todetermine if MAP kinase activation is impaired, cells are exposed toagonists or antagonists for the periods of time described previouslythen cell lysates prepared as described previously (Maile L A andClemmons D R Circ Res, 93: 925-931 (2003)). 30 μl of cell lysate isanalyzed directly by SDS-PAGE with immunoblotting for the phosphorylatedform of ERK 1/2 (an indication of MAP kinase activity) (Ling Y, Maile LA, Clemmons D R, Mol Endocrinol, 17:1824-1833 (2003)). It would beanticipated that the time course intensity of MAP kinase activation willbe prolonged by β3 agonists and inhibited by β3 agonists.

Cell Replication.

Smooth muscle and/or endothelium are plated at relatively low density,10⁴/cm² in low serum (0.2%) containing medium. 24 hr after plating,cells are quiesced in 0.2% platelet poor plasma containing medium. 24 hrlater the cultures are exposed to increasing concentrations of IGF-Ibetween 0 and 100 ng/ml and the β3 agonists or antagonists. After 48 hr,the cell cultures are stained with trypan blue and the cell number isdetermined by manual counting. If β3 is occupied by an agonist thenthere is at least a 2 fold increase in cell number over this timeperiod. Whereas if an antagonist is added there is often less than 20%increase in cell number.

Cell Migration.

Confluent quiescent smooth muscle or endothelial cell cultures arewounded with a razor blade as described in Maile L A, Imai Y, Clarke JB, Clemmons D R, J. Biol. Chem, 277:1800-1805 (2002). The wounds areexamined to determine that a straight edge has been obtained and thereare no grooves in the plate. At least five areas that are correctlywounded are then identified with a color marker. IGF-I is added atconcentrations of either 50 or 100 ng/ml and various concentrations ofthe angonists or antagonists are added to at least duplicate cultures.After 72 hr the number of cells that have migrated at least 50 micronsfrom the wound edge are determined and counted following staining withmethylene blue. IGF-I normally stimulates between 20 and 50 cells permicroscopic field to migrate this distance. In the presence of anantagonist, there is generally fewer than 5 cells/microscopic field thatmigrate but agonists may increase the response to IGF-I by as much as 2fold.

The present invention is explained in greater detail in the followingnon-limiting Examples.

Example 1 DNA Synthesis

Cells are plated at a density of 2.5×10⁴/cm² in 96-well tissue cultureplates and grown for 5 days without a medium change. They were rinsedonce with serum-free DMEM and serum starved by incubating with DMEM plus0.2% platelet poor plasma (PPP) for 24 h. The cells are then exposed toIGF-I plus any treatment and incubated at 37 C for 24 h, and the amountof [³H]thymidine incorporated into DNA was determined as described in:Imai Y and Clemmons D R. Roles of Phosphatidylinositol 3-Kinase andMitogen-Activated Protein Kinase Pathways in Stimulation of VascularSmooth Muscle Cell Migration and Deoxyribonucleic Acid Synthesis byInsulin-Like Growth Factor-I. Endocrinology 140, 4228-4235 (1999).

Example 2 Peptide Synthesis

Peptides are synthesized using FMOC chemistry on a Rainin MultiplePeptide synthesizer. Activation of FMOC amino acids and acylationutilizes HATU in the presence of a base (N methyl morpholine). Uponcompletion of acylation, the FMOC protecting group is removed with 20%piperidine in dimethylformamide. After synthesis, the peptide is removedfrom the resin and deprotected by treatment with 95% trifluoroaceticacid containing appropriate organic scavengers.

Cleaved, deprotected peptides are precipitated in cold ether,resuspended in a dilute TFA/acetonitrile mix, and purified by highperformance liquid chromatography on a reverse phase resin with anincreasing acetonitrile gradient.

Quality control of the peptide product is assessed by analytical HPLCand by matrix assisted laser desorption ionization time-of-flight massspectrometry. Purified peptide is lyophilized and stored at −20° C.

Example 3 Identification of Agonists

As noted above, the agonists bind to a specific region on the αVβ3integrin receptor that has not been identified previously as a regionthat would result in receptor activation. All agonists that we havedetermined to bind in this region contain a region of sequence that iscommonly termed a heparin binding domain. This heparin binding domain ispresent in 5 ligands that we have found to date that bind to this regionof αVβ3. The documentation that they bind to a specific region of αVβ3has been provided by two types of experiments. First we have apolyclonal antibody that is known to react with this region of αVβ3,which is the region of the β3 subunit that is located between aminoacids at positions 177 and 183. When this polyclonal antibody isincubated with any of the known agonists, it completely inhibits theirability to bind.

A more definitive experiment was conducted in which the identical regionof the β1 integrin was substituted by mutagenesis for this 8 amino acidregion within β3. The mutant protein was then expressed in CHO cellswhich do not constitutively express β3. Binding assays were thenconducted using these ligands. It was shown that this mutation resultedin a complete loss of binding and an inability to activate β3.Activation was measured by β3 phosphorylation which has been shown to bestimulated by binding of these agonists to this region of β3. These dataindicate this is the region of β3 that binds to these ligands.

Example 4 Identification of Antagonist

A synthetic peptide bearing the following structure: KKQRFRHL (SEQ IDNO: 6) was found to have inhibitory activity in each of these biologicassays. Activation of the IGF-I receptor by IGF-I could be inhibited byapproximately 60%. Furthermore analysis has shown that a cyclizedpeptide containing this sequence is a more potent inhibitor.Substitution of a hydrophobic residue for arginine at position 208results in the greatest loss of activity.

Example 5 Additional Agonists

Additional peptide agonists that can be used to carry out the presentinvention are set forth in Table 1 below. Such peptide agonists aresynthesized as described in Example 1 above.

TABLE 1 Examples of additional IGF-I agonist peptides.Agonist (X_(m)BBXXABBBX_(n))* (SEQ ID NO: 7) KKQRFRHR ( SEQ ID NO: 8)RKQRFRHR (SEQ ID NO: 9) KRQRFRHR (SEQ ID NO: 10)KKQRFKHR (SEQ ID NO: 11) KKQRFRHK (SEQ ID NO: 12)KKQRFRRR (SEQ ID NO: 13) KKQRFRKR (SEQ ID NO: 14)KKQRFRHR (SEQ ID NO: 15) HKQRFRHR (SEQ ID NO: 16)KHQRFRHR (SEQ ID NO: 17) RRQRFRHR (SEQ ID NO: 18)KKQRFKHK (SEQ ID NO: 19) KKQKFRHR (SEQ ID NO: 20)KKNRFRHR (SEQ ID NO: 21) KKQRYRHR (SEQ ID NO: 22)KKQRWRHR (SEQ ID NO: 23) KKQRHRHR (SEQ ID NO: 24)AKKQRFRHRN (SEQ ID NO: 25) LAKKQRFRHRNR (SEQ ID NO: 26)SLAKKQRFRHRNRK (SEQ ID NO: 27) PSLAKKQRFRHRNRKG (SEQ ID NO: 28)RPSLAKKQRFRHRNRKG (SEQ ID NO: 29) AKKQRFRHR (SEQ ID NO: 30)LAKKQRFRHR (SEQ ID NO: 31) SLAKKQRFRHR (SEQ ID NO: 32)PSLAKKQRFRHR (SEQ ID NO: 33) RPSLAKKQRFRHR (SEQ ID NO: 34)KKQRFRHRN (SEQ ID NO: 35) KKQRFRHRNR (SEQ ID NO: 36)KKQRFRHRNRK (SEQ ID NO: 37) KKQRFRHRNRKG (SEQ ID NO: 38)RPSLAKKQRFRHRN (SEQ ID NO: 39) RPSLAKKQRFRHRNR (SEQ ID NO: 40)RPSLAKKQRFRHRNRK (SEQ ID NO: 41) AKKQRFRHRNRKG (SEQ ID NO: 42)LAKKQRFRHRNRKG (SEQ ID NO: 43) SLAKKQRFRHRNRKG (SEQ ID NO: 44) *B =basic amino acid (R, K, H); X = any amino acid; A = aromatic amino acid(F, Y, W, H). m = any integer from 0-5, n = any integer from 0-4.

Example 6 Additional Antagonists

Additional peptide antagonists that can be used to carry out the presentinvention are set forth in Table 2 below. Such peptide agonists aresynthesized as described in Example 2 above.

TABLE 2 Examples of IGF-I antagonist peptides.Antagonist (X_(m)BBXXABBHX_(n))* (SEQ ID NO: 45)KKQRFRHL (SEQ ID NO: 46) KKQRFRHA (SEQ ID NO: 47)RKGRYKRA (SEQ ID NO: 48) KKQRFRHI (SEQ ID NO: 49)KKQRFRHV (SEQ ID NO: 50) KKQRFRHM (SEQ ID NO: 51)KKQRFRHC (SEQ ID NO: 52) KKQRFRHF (SEQ ID NO: 53)KKQRFRHW (SEQ ID NO: 54) RKQRFRHL (SEQ ID NO: 55)RKQRFRHI (SEQ ID NO: 56) RKQRFRHV (SEQ ID NO: 57)RKQRFRHM (SEQ ID NO: 58) RKQRFRHC (SEQ ID NO: 59)RKQRFRHF (SEQ ID NO: 60) RKQRFRHW (SEQ ID NO: 61)AKKQRFRHLN (SEQ ID NO: 62) LAKKQRFRHLNR (SEQ ID NO: 63)SLAKKQRFRHLNRK (SEQ ID NO: 64) PSLAKKQRFRHLNRKG (SEQ ID NO: 65)RPSLAKKQRFRHLNRKG (SEQ ID NO: 66) AKKQRFRHL (SEQ ID NO: 67)LAKKQRFRHL (SEQ ID NO: 68) SLAKKQRFRHL (SEQ ID NO: 69)PSLAKKQRFRHL (SEQ ID NO: 70) RPSLAKKQRFRHL (SEQ ID NO: 71)KKQRFRHLN (SEQ ID NO: 72) KKQRFRHLNR (SEQ ID NO: 73)KKQRFRHLNRK (SEQ ID NO: 74) KKQRFRHLNRKG (SEQ ID NO: 75)RPSLAKKQRFRHLN (SEQ ID NO: 76) RPSLAKKQRFRHLNR (SEQ ID NO: 77)RPSLAKKQRFRHLNRK (SEQ ID NO: 78) AKKQRFRHLNRKG (SEQ ID NO: 79)LAKKQRFRHLNRKG (SEQ ID NO: 80) SLAKKQRFRHLNRKG (SEQ ID NO: 81) *B =basic amino acid (R, K, H); X = any amino acid; A = aromatic amino acid(F, Y, W, H) and H = hydrophobic amino acid (I, L, V, M, F, W, C). H(when referring to hydrophobic amino acid and not referring tohistidine) may also be phospho-serine(S-PO₄). m = any integer from 0-5,n = any integer from 0-4.

Example 7 Preparation of Peptide for Immunization

In order to prepare an antibody, the sequence of the β3 subunit thatbinds to the heparin binding domain of vitronectin and other ligands, asynthetic peptide was prepared. The peptide was synthesized using FMOCchemistry using a Rainin multiple peptide synthesizer. Activation ofFMCO amino acids and acylation utilizes HTAU in the presence of a base(n-methylmorpholine). Upon completion of acylation, the FMOC protectinggroup is removed with 20% piperidine in dimethylformamide. Aftersynthesis, the peptide is removed from the resin and deprotected bytreatment with 95% triflouroacetic acid containing appropriate organicscavengers. Cleaved and deprotected peptide was then precipitated withcold ether and resuspended in dilute TFA/acetonitrile and purified byhigh performance liquid chromatography on a reverse phase resin andeluted with an increasing acetonitrile gradient. The quality of thepeptide product was assessed by analytical HPLC and by matrix assistedlaser desorption ionization time-of-flight mass spectrometry. Thepurified peptide was then lyophylized and stored at −20 C. The mass ofeluting peptide was verified as containing the correct amino acids bycomparison to the known masses in the database.

Conjugation of the β3 cysteine loop peptide (CYDMKTTC) (SEQ ID NO: 82)to Imject Maleimide Activated Mariculture Keyhole Limpet Hemocyanin(Pierce, Rockford, Ill.). 0.7 mg, 1.2 mg and 2.1 mg amounts of peptidewere weighed and each was dissolved separately before addition to KLH in500 mcl of 0.03 M NaH₂ PO₄, pH 7.2 containing 0.9 M sodium chloride. 2mg of maleimide activated KLH was dissolved in 500 mcl of distilledwater. 0.7 mg of dissolved peptide was then added to the KLH solutionand incubated at room temperature for 20 min. An additional tubecontaining 0.7 mg of peptide dissolved in buffer and added to the sameKLH solution was incubated again 25 min at room temperature. 1.2 mg and2.1 mg of peptide were sequentially added to the KLH solution andfurther incubated for 1 hr intervals after each addition, at roomtemperature. The peptide conjugate was removed and dialyzed 24 hragainst 21 of 0.083 M sodium H₂PO₄ pH 7.2, 0.9 M NaCl with one exchange.The total 4.7 mg of peptide/KLH conjugate was divided into 5 equalaliquots, lyophylized and frozen at −20° C. for later use.

Example 8 Rabbit Immunization

New Zealand white rabbit females between 2 and 3 months of age were usedfor immunization. One of the 5 lyophylized aliquots containing 1.34 mgof conjugate was dissolved in 450 mcl of distilled water. 450 mcl ofcomplete Freunds adjuvant was added and the mixture homogenized. 10intradermal injections containing between 40-50 ul of mixture perinjection were utilized and injected into the rabbits back. After aninterval between 4 and 5 weeks the animal was bled and 9-11 ml of wholeblood was removed. The animal then received a booster injectioncontaining 1.3 mg of conjugate dissolved in 450 mcl of distilled watercontaining 450 ul of incomplete Freunds adjuvant. This mixture wasinjected at one site subcutaneously. The rabbit was then bled monthlyvia the central ear artery, collecting 9-12 ml at each bleed. Theimmunizations were repeated 4 times and bleeding conducted at 4-5 weekintervals.

Example 9 Antibody Purification

The antibody was purified over a protein G affinity column that had beenpurchased from Sigma. 10-15 ml of crude rabbit serum was diluted with anequal volume of 25 mM sodium H₂PO₄, pH 7.2 and passed multiple timesover a protein G affinity column previously equilibrated in the samebuffer for 16 hrs at 4° C. The column was then eluted with 10 columnvolumes (100 ml total) of 25 mM sodium phosphate 7.2 and the IgG elutedwith 0.1 M glycine HCL, pH 2.7. The chromatographic fractions containingthe antibody were neutralized with 1.0 M TRIS, pH 9 to pH 7.2 anddialyzed against 25 mM sodium phosphate, ph 7.2 containing 50 mM sodiumchloride. After dialysis the protein G purified antibody was stored −20°C.

Example 10 Preparation of Affinity Column and Antibody Purification

In order to purify the antibody from whole serum, a peptide affinitycolumn was prepared that contained the exact amino acid sequence ofimmunogen. The cysteine loop peptide (CYDMKTTC) (SEQ ID NO: 82) wascoupled to agarose using Sulfolink coupling gel (Pierce Chemical Co.). 5ml of coupling gel was equilibrated with a buffer containing 50 mM TrispH 8.5, 5 mM sodium ETDA. 0.7 mg of the synthetic peptide was dissolvedin 1 ml of coupling buffer and added to 5 ml of gel and incubated atroom temperature with mixing for 30 min. The procedure was repeated with0.8 and 2 mg aliquots of peptide added sequentially to the same tubecontaining the coupling gel and incubated for 30 min. During the laststep 3.2 mg of peptide in the coupling buffer was added and the wholemixture reincubated for an additional 3 hr. After the final 3 hrincubation, the material was spun at 1000×g and the supernatant removed.5 ml of 50 M cysteine in the above described buffer was added to thecoupling gel and the incubation continued at room temperature for 1 hrwith gentle mixing to block unreacted sites. The gel was stored in 0.25%sodium azide in distilled water until use. To purify the antibody theaffinity column was first equilibrated with 50 mM Tris pH 7.2containing, 50 mM sodium chloride. The antibody pool that had beeneluted from the protein G affinity column and previously dialyzed wascirculated over the column for 36 hr. The column was washed with 10column volumes (100 ml) of the loading buffer and eluted with 0.1 Mglycine, pH 2.7. The antibody was then neutralized with 1 M Tris, pH 9and stored at −20° C. until use. The final protein concentration was 180mcg/ml.

Example 11 Production and Screening of Monoclonal Antibodies

Immunization.

Pathogen-free Swiss Webster mice are utilized for immunization. Theconjugated peptide described above is mixed with emulsified mouse RIBI(MPL+TDDTM emulsion) adjuvant and 300 mcg of the emulsified antigeninjected intraperitoneally. The injections are repeated at three weekintervals, twice. Antibody titers are determined by withdrawing 50-100mcl of blood from the tail vein at these three week intervals. The titeris determined by testing the reactivity of the mouse serum forimmobilized β3 antigen. In mice where sufficient antibody titer isobtained after six weeks the mice are sacrificed and the spleens andlymph nodes removed for fusion to myeloma cells for hybridoma formation.

Hybridoma Formation.

Two mice are selected for spleen harvest. These mice are boosted a thirdtime with 300 mcg of antigen then four days later sacrificed. Blood andspleen are collected. Spleen cells will be harvested and fused with63-AGA.65 (ATCCCRL-1580) cells using a 50% PEG solution. These fusedcells are then plated in a 96 well plate at 1×10⁵ cells per well in HATselection medium. After 12-14 days the fusion plates or clones are fedin HT media. At this time medium is collected for screening by ELIZAassay as described below to identify desired hybridoma cells.

ELISA Materials.

The ELISA is carried out with the following materials:

96 well Immulon IV plates (Fisher Cat #14-245-153)

1× coating buffer (0.05M carbonate/bicarbonate buffer pH 9.6 Sigma Cat #C3041)

2% BSA in PBS (Blocking Buffer)

0.5% BSA in PBS (ELISA Buffer)

0.05% Tween PBS (Wash buffer)

DEA developer

The DEA Developer (for 500 ml): is produced from 4.8 ml of 85%Diethanolamine (Fisher Cat # D45); 0.25 ml of 1M MgCl₂; and pNPP tablets(Sigma Cat # N2765). To prepare the developer, dissolve DEA in 400 ml ofsterile water and adjust pH to 10 with HCL and NaOH. Add MgCl₂. Bringvolume up to 500 ml. Store at 4° C. Wrap in foil to protect from light.Immediately before use add 1 tablet of pNPP to 20 ml of buffer

The Secondary Antibody is goat anti mouse IgG alkaline phosphataseconjugate (Jackson Immunoresearch Cat #115-055-164)

Peptide at 1 mg/ml in PBS.

ELISA Method. With materials prepared as described above, ELISAscreening of monoclonal antibodies produced as described above iscarried out to isolate and provide a monoclonal antibody of the presentinvention as follows:

-   1. Coat plates with 50 μl/well of peptide in coating buffer at    concentration of 5 μg/ml at 4° C. overnight.-   2. Wash plates with 0.05% Tween-   3. Block plates with 200 μl/well blocking buffer overnight at 4° C.-   4. Repeat step 2-   5. Add primary antibody (test antibody) at 40 μl/well (Supernatants)    or 50 μl/well (serum dilutions) and incubate for 1 hour at room    temperature-   6. Wash plates with 0.05% Tween in PBS-   7. Add 50 μl/well of secondary antibody at 1:2000 dilution and    incubate at room temperature for 1 hour-   8. Wash plates with 0.05% Tween in PBS-   9. Add 50 μl/well of DEA developer and allow to incubate-   10. Read in spectrophotometer at 405 nM.

Hybridoma LAM-CLOOP 101, which produces an antibody of the presentinvention, was produced by the methods described above.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A method of inhibiting cell division of anendothelial cell, comprising contacting the endothelial cell with aneffective amount of an antibody that: (a) specifically binds to thecysteine loop domain at amino acids 177 to 184 of a human β3 integrin,wherein said amino acids consist of SEQ ID NO: 82; (b) does notspecifically bind to the RGD binding site of a human β3 integrin; and(c) specifically binds to the cysteine loop domain at amino acids 177 to184 of a pig β3 integrin, wherein said amino acids consist of SEQ ID NO:82, thereby inhibiting cell division of the endothelial cell.
 2. Themethod of claim 1, wherein the antibody is coupled to a detectablegroup.
 3. The method of claim 1, wherein the antibody is coupled to atherapeutic group.
 4. The method of claim 1, wherein the antibody is amonoclonal antibody.
 5. The method of claim 1, wherein the antibody doesnot specifically inhibit ligand occupancy of the RGD binding site of theβ3 integrin.
 6. The method of claim 1, wherein the antibody does notstimulate the specific biochemical events that are stimulated bypeptides that bind to the RGD domain binding site of human β3 integrin.7. A method of inhibiting vitronectin occupancy of a human β3 integrin,comprising contacting an endothelial cell comprising a human β3 integrinin the presence of vitronectin with an effective amount of an antibodythat: (a) specifically binds to the cysteine loop domain at amino acids177 to 184 of a human β3 integrin, wherein said amino acids consist ofSEQ ID NO: 82; (b) does not specifically bind to the RGD binding site ofa human β3 integrin; and (c) specifically binds to the cysteine loopdomain at amino acids 177 to 184 of a pig β3 integrin, wherein saidamino acids consist of SEQ ID NO: 82, thereby inhibiting vitronectinoccupancy of the human β3 integrin.
 8. The method of claim 7, whereinthe antibody is coupled to a detectable group.
 9. The method of claim 7,wherein the antibody is coupled to a therapeutic group.
 10. The methodof claim 7, wherein the antibody is a monoclonal antibody.
 11. Themethod of claim 7, wherein the antibody does not specifically inhibitligand occupancy of the RGD binding site of the β3 integrin.
 12. Themethod of claim 7, wherein the antibody does not stimulate the specificbiochemical events that are stimulated by peptides that bind to the RGDdomain binding site of human β3 integrin.